Large language models (LLMs) have shown significant potential to improve diagnostic performance for clinical professionals.Existing multi-agent paradigms rely mainly on prompt engineering, suffering from improper agent selection and insufficient knowledge integration.In this work, we propose a novel framework KACR (Knowledge-Aware Co-Reasoning) that integrates structured knowledge reasoning into multidisciplinary collaboration from two aspects: (1) a reinforcement learning-optimized agent that uses clinical knowledge graphs to guide dynamic discipline determination; (2) a multidisciplinary collaboration strategy that enables robust consensus through integration of domain-specific expertise and interdisciplinary persuasion mechanism.Extensive experiments conducted on both academic and real-world datasets demonstrate the effectiveness of our method.1 Main work done when working at Alibaba: https:// anonymous.4open.science/r/KACR_RL-2B64agents iteratively refine their positions through evidence-based persuasion.Persuasion strength is explicitly measured using the value estimates from the Critic trained in Step I, ensuring that consensus-building is aligned with the underlying clinical knowledge structure.This dual mechanism effectively balances specialized expertise with collective intelligence during differential diagnosis.Knowledge Graph.The clinical knowledge graph (CKG), denoted as G, comprises three core components: entity set V, structural relation set E, and relation type set R. The entities are classified into three distinct categories: symptom entities V s , disease entities V d , and discipline entities V c .Each relation is formally represented as a triplet (v i , r, v j ), where v i (head entity) and v j (tail entity) are interconnected through the relation type r ∈ R.

The significant advances in computing power have led to a surge in model complexity. Training such models today increasingly relies on transfer learning, where models are pre-trained on large datasets and later fine-tuned for different domains, allowing the knowledge in the pre-trained model to be effectively reused and customized for these specific domains. However, such a learning paradigm also opens new attack surfaces on the fine-tuned model. Particularly, a privacy risk never studied before is the threat posed by the adversary affecting the pre-training process to the downstream user's private data for fine-tuning the model: A manipulated pre-trained model can render its fine-tuned version vulnerable to privacy attacks, such as membership inference attacks (MIAs) where the presence of a given sample in the fine-tuning dataset can be determined by querying the vulnerable model. A unique challenge in understanding this privacy risk is how to amplify the membership leakage while ensuring the performance of the fine-tuned model. To address this challenge, we introduce a new technique - active robustness overfitting (ARO). This approach actively induces robustness overfitting during pre-training, which amplifies membership leakage in the downstream task without affecting its accuracy, while also maintaining the stealthiness of the attack. Our extensive evaluations across various datasets and diverse MIA scenarios demonstrate that our methods can effectively amplify membership leakage while preserving satisfactory downstream test accuracy, which contributes to a better understanding of the privacy risk introduced by transfer learning.

MOTIVATION: As the SARS-CoV-2 virus rapidly evolves, predicting the trajectory of viral mutations has become a critical yet complex task. A deep understanding of future mutation patterns, in particular the mutations that will prevail in the near future, is vital in steering diagnostics, therapeutics, and vaccine strategies for disease control. RESULTS: In this study, we developed a model to forecast future SARS-CoV-2 mutation surges in real-time, using historical mutation frequency data from the USA. We transformed the temporal prediction problem into a supervised learning framework using a sliding window approach. This involved breaking the time series of mutation frequencies into very short segments. Considering the time-dependent nature of the data, we focused on modeling the first-order derivative of the mutation frequency. We predicted the final derivative in each segment based on the preceding derivatives, employing various machine learning methods, including random forest, XGBoost, support vector machine, and neural network models. Empowered by the novel transformation strategy and the high capacity of machine learning models, we observed low prediction error that is confined within 0.1% and 1% when making predictions of mutation rates for the future 30 and 80 days, respectively. In addition, the method also led to a notable increase in prediction accuracy compared to traditional time-series models, as evidenced by much lower MAE (Mean Absolute Error) and MSE (Mean Squared Error) for predictions made within different time horizons. To further assess the method's effectiveness and robustness in predicting mutation patterns for unforeseen mutations, we first designed a synthetic case where we categorized all mutations into three major patterns. The model demonstrated its robustness by accurately predicting unseen mutation patterns when training on data from two pattern categories while testing on the third pattern category, showcasing its potential in forecasting a variety of mutation trajectories. We then applied our method to prediction for a recent time frame between 1 January 2025 and 10 June 2025, for both the USA and UK, where the model training was conducted using frequency sequence data collected between 12 December 2019 and 26 January 2023 in the USA. The model demonstrated superior performance for both datasets. AVAILABILITY AND IMPLEMENTATION: To enhance accessibility and utility, we built our methodology into a GitHub package (https://github.com/ZhouXY199502/SWD). Our method has the potential applicability to study other infectious diseases or forecasting tasks, thus extending its relevance beyond the current COVID pandemic.

The rapid advancements of large language models (LLMs) have raised public concerns about the privacy leakage of personally identifiable information (PII) within their extensive training datasets. Recent studies have demonstrated that an adversary could extract highly sensitive privacy data from the training data of LLMs with carefully designed prompts. However, these attacks suffer from the model's tendency to hallucinate and catastrophic forgetting (CF) in the pre-training stage, rendering the veracity of divulged PIIs negligible. In our research, we propose a novel attack, Janus, which exploits the fine-tuning interface to recover forgotten PIIs from the pre-training data in LLMs. We formalize the privacy leakage problem in LLMs and explain why forgotten PIIs can be recovered through empirical analysis on open-source language models. Based upon these insights, we evaluate the performance of Janus on both open-source language models and two latest LLMs, i.e., GPT-3.5-Turbo and LLaMA-2-7b. Our experiment results show that Janus amplifies the privacy risks by over 10 times in comparison with the baseline and significantly outperforms the state-of-the-art privacy extraction attacks including prefix attacks and in-context learning (ICL). Furthermore, our analysis validates that existing fine-tuning APIs provided by OpenAI and Azure AI Studio are susceptible to our Janus attack, allowing an adversary to conduct such an attack at a low cost.

MOTIVATION: Tandem mass spectrometry (MS/MS) is a crucial technology for large-scale proteomic analysis. The protein database search or the spectral library search are commonly used for peptide identification from MS/MS spectra, which, however, may face challenges due to experimental variations between replicated spectra and similar fragmentation patterns among distinct peptides. To address this challenge, we present SpecEncoder, a deep metric learning approach to address these challenges by transforming MS/MS spectra into robust and sensitive embedding vectors in a latent space. The SpecEncoder model can also embed predicted MS/MS spectra of peptides, enabling a hybrid search approach that combines spectral library and protein database searches for peptide identification. RESULTS: We evaluated SpecEncoder on three large human proteomics datasets, and the results showed a consistent improvement in peptide identification. For spectral library search, SpecEncoder identifies 1%-2% more unique peptides (and PSMs) than SpectraST. For protein database search, it identifies 6%-15% more unique peptides than MSGF+ enhanced by Percolator, Furthermore, SpecEncoder identified 6%-12% additional unique peptides when utilizing a combined library of experimental and predicted spectra. SpecEncoder can also identify more peptides when compared to deep-learning enhanced methods (MSFragger boosted by MSBooster). These results demonstrate SpecEncoder's potential to enhance peptide identification for proteomic data analyses. AVAILABILITY AND IMPLEMENTATION: The source code and scripts for SpecEncoder and peptide identification are available on GitHub at https://github.com/lkytal/SpecEncoder. Contact: [email protected].

Xurui Li, Yue Qin, Rui Zhu, Tianqianjin Lin, Yongming Fan, Yangyang Kang, Kaisong Song, Fubang Zhao, Changlong Sun, Haixu Tang, Xiaozhong Liu. Proceedings of the 2023 Conference on Empirical Methods in Natural Language Processing. 2023.

The extensive applications of deep neural network (DNN) and its increasingly complicated architecture and supply chain make the risk of backdoor attacks more realistic than ever. In such an attack, the adversary either poisons the training data of a DNN model or manipulates its training process to stealthily inject a covert backdoor task, alongside the primary task, so as to strategically misclassify inputs carrying a trigger. Defending against such an attack, particularly removing the backdoor effect from an infected model, is known to be hard. For this purpose, prior research either requires a recovered trigger, which is hard to come by, or attempts to fine-tune a model on its primary task, which becomes less effective when the clean data is scarce. In this paper, we present a simple yet surprisingly effective technique to induce "selective amnesia" on a backdoored model. Our approach, called SEAM, has been inspired by the problem of catastrophic forgetting (CF), a long standing issue in continual learning. Our idea is to retrain a given DNN model on randomly labeled clean data, to induce a CF on the model, leading to a sudden forget on both primary and backdoor tasks; then we recover the primary task by retraining the randomized model on correctly labeled clean data. We analyzed SEAM by modeling the unlearning process as continual learning and further approximating a DNN using Neural Tangent Kernel for measuring CF. Our analysis shows that our random-labeling approach actually maximizes the CF on an unknown backdoor in the absence of triggered inputs, and also preserves some feature extraction in the network to enable a fast revival of the primary task. We further evaluated SEAM on both image processing and Natural Language Processing tasks, under both data contamination and training manipulation attacks, over thousands of models either trained on popular image datasets or provided by the TrojAI competition. Our experiments show that SEAM vastly outperforms the state-of-the-art unlearning techniques, achieving a high Fidelity (measuring the gap between the accuracy of the primary task and that of the backdoor) efficiently (e.g., about 30 times faster than training a model from scratch on the MNIST dataset), with only a small amount of clean data (e.g., with a size of just 0.1% of training data for TrojAI models).

MOTIVATION: Tandem mass spectrometry is an essential technology for characterizing chemical compounds at high sensitivity and throughput, and is commonly adopted in many fields. However, computational methods for automated compound identification from their MS/MS spectra are still limited, especially for novel compounds that have not been previously characterized. In recent years, in silico methods were proposed to predict the MS/MS spectra of compounds, which can then be used to expand the reference spectral libraries for compound identification. However, these methods did not consider the compounds' 3D conformations, and thus neglected critical structural information. RESULTS: We present the 3D Molecular Network for Mass Spectra Prediction (3DMolMS), a deep neural network model to predict the MS/MS spectra of compounds from their 3D conformations. We evaluated the model on the experimental spectra collected in several spectral libraries. The results showed that 3DMolMS predicted the spectra with the average cosine similarity of 0.691 and 0.478 with the experimental MS/MS spectra acquired in positive and negative ion modes, respectively. Furthermore, 3DMolMS model can be generalized to the prediction of MS/MS spectra acquired by different labs on different instruments through minor fine-tuning on a small set of spectra. Finally, we demonstrate that the molecular representation learned by 3DMolMS from MS/MS spectra prediction can be adapted to enhance the prediction of chemical properties such as the elution time in the liquid chromatography and the collisional cross section measured by ion mobility spectrometry, both of which are often used to improve compound identification. AVAILABILITY AND IMPLEMENTATION: The codes of 3DMolMS are available at https://github.com/JosieHong/3DMolMS and the web service is at https://spectrumprediction.gnps2.org.

Concerns regarding inappropriate leakage of sensitive personal information as well as unauthorized data use are increasing with the growth of genomic data repositories. Therefore, privacy and security of genomic data have become increasingly important and need to be studied. With many proposed protection techniques, their applicability in support of biomedical research should be well understood. For this purpose, we have organized a community effort in the past 8 years through the integrating data for analysis, anonymization and sharing consortium to address this practical challenge. In this article, we summarize our experience from these competitions, report lessons learned from the events in 2020/2021 as examples, and discuss potential future research directions in this emerging field.

Trusted execution environments (TEE) such as Intel's Software Guard Extension (SGX) have been widely studied to boost security and privacy protection for the computation of sensitive data such as human genomics. However, a performance hurdle is often generated by SGX, especially from the small enclave memory. In this paper, we propose a new Hybrid Secured Flow framework (called "HySec-Flow") for large-scale genomic data analysis using SGX platforms. Here, the data-intensive computing tasks can be partitioned into independent subtasks to be deployed into distinct secured and non-secured containers, therefore allowing for parallel execution while alleviating the limited size of Page Cache (EPC) memory in each enclave. We illustrate our contributions using a workflow supporting indexing, alignment, dispatching, and merging the execution of SGX- enabled containers. We provide details regarding the architecture of the trusted and untrusted components and the underlying Scorn and Graphene support as generic shielding execution frameworks to port legacy code. We thoroughly evaluate the performance of our privacy-preserving reads mapping algorithm using real human genome sequencing data. The results demonstrate that the performance is enhanced by partitioning the time-consuming genomic computation into subtasks compared to the conventional execution of the data-intensive reads mapping algorithm in an enclave. The proposed HySec-Flow framework is made available as an open-source and adapted to the data-parallel computation of other large-scale genomic tasks requiring security and scalable computational resources.

A trusted execution environment (TEE) such as Intel Software Guard Extension (SGX) runs attestation to prove to a data owner the integrity of the initial state of an enclave, including the program to operate on her data. For this purpose, the data-processing program is supposed to be open to the owner or a trusted third party, so its functionality can be evaluated before trust being established. In the real world, however, increasingly there are application scenarios in which the program itself needs to be protected (e.g., proprietary algorithm). So its compliance with privacy policies as expected by the data owner should be verified without exposing its code. To this end, this paper presents Deflection, a new model for TEE-based delegated and flexible in-enclave code verification. Given that the conventional solutions do not work well under the resource-limited and TCB-frugal TEE, we come up with a new design inspired by Proof-Carrying Code. Our design strategically moves most of the workload to the code generator, which is responsible for producing easy-to-check code, while keeping the consumer simple. Also, the whole consumer can be made public and verified through a conventional attestation. We implemented this model on Intel SGX and demonstrate that it introduces a very small part of TCB. We also thoroughly evaluated its performance on micro- and macro- benchmarks and real-world applications, showing that the design only incurs a small overhead when enforcing several categories of security policies.

In the past decade, the security of cellular networks has been increasingly under scrutiny, leading to the discovery of numerous vulnerabilities that expose the network and its users to a wide range of security risks, from denial of service to information leak. However, most of these findings have been made through ad-hoc manual analysis, which is inadequate for fundamentally enhancing the security assurance of a system as complex as the cellular network. An important observation is that the massive amount of technical documentation of cellular network can provide key insights into the protection it puts in place and help identify potential security flaws. Particularly, we found that such documentation often contains hazard indicators (HIs) – the statement that describes a risky operation (e.g., abort an ongoing procedure) when a certain event happens at a state, which can guide a test on the system to find out whether the operation can indeed be triggered by an unauthorized party to cause harm to the cellular core or legitimate users’ equipment. Based upon this observation, we present in this paper a new framework that makes the first step toward intelligent and systematic security analysis of cellular networks. Our approach, called Atomic, utilizes natural-language processing and machine learning techniques to scan a large amount of LTE documentation for HIs. The HIs discovered are further parsed and analyzed to recover state and event information for generating test cases. These test cases are further utilized to automatically construct tests in an LTE simulation environment, which runs the tests to detect the vulnerabilities in the LTE that allow the risky operations to happen without proper protection. In our research, we implemented Atomic and ran it on the LTE NAS specification, including 549 pages with 13,598 sentences and 283,850 words. In less than 5 hours, our prototype reported 42 vulnerabilities from 192 HIs discovered, including 10 never reported before, under two threat models. All these vulnerabilities have been confirmed through end-to-end attacks, which lead to unauthorized disruption of the LTE service a legitimate user’s equipment receives. We reported our findings to authorized parties and received their confirmation that these vulnerabilities indeed exist in major commercial carriers and $2,000 USD reward from Google.

Domain Adaptation (DA) targets at adapting a model trained over the well-labeled source domain to the unlabeled target domain lying in different distributions. Existing DA normally assumes the well-labeled source domain is class-wise balanced, which means the size per source class is relatively similar. However, in real-world applications, labeled samples for some categories in the source domain could be extremely few due to the difficulty of data collection and annotation, which leads to decreasing performance over target domain on those few-shot categories. To perform fair cross-domain adaptation and boost the performance on these minority categories, we develop a novel Generative Few-shot Cross-domain Adaptation (GFCA) algorithm for fair cross-domain classification. Specifically, generative feature augmentation is explored to synthesize effective training data for few-shot source classes, while effective cross-domain alignment aims to adapt knowledge from source to facilitate the target learning. Experimental results on two large cross-domain visual datasets demonstrate the effectiveness of our proposed method on improving both few-shot and overall classification accuracy comparing with the state-of-the-art DA approaches.

MOTIVATION: The availability of human genomic data, together with the enhanced capacity to process them, is leading to transformative technological advances in biomedical science and engineering. However, the public dissemination of such data has been difficult due to privacy concerns. Specifically, it has been shown that the presence of a human subject in a case group can be inferred from the shared summary statistics of the group, e.g. the allele frequencies, or even the presence/absence of genetic variants (e.g. shared by the Beacon project) in the group. These methods rely on the availability of the target's genome, i.e. the DNA profile of a target human subject, and thus are often referred to as the membership inference method. RESULTS: In this article, we demonstrate the haplotypes, i.e. the sequence of single nucleotide variations (SNVs) showing strong genetic linkages in human genome databases, may be inferred from the summary of genomic data without using a target's genome. Furthermore, novel haplotypes that did not appear in the database may be reconstructed solely from the allele frequencies from genomic datasets. These reconstructed haplotypes can be used for a haplotype-based membership inference algorithm to identify target subjects in a case group with greater power than existing methods based on SNVs. AVAILABILITY AND IMPLEMENTATION: The implementation of the membership inference algorithms is available at https://github.com/diybu/Haplotype-based-membership-inferences.

Membership Inference Attacks (MIAs) aim to determine the presence of a record in a machine learning model's training data by querying the model. Recent work has demonstrated the effectiveness of MIA on various machine learning models and corresponding defenses have been proposed. However, both attacks and defenses have focused on an adversary that indiscriminately attacks all the records without regard to the cost of false positives or negatives. In this work, we revisit membership inference attacks from the perspective of a pragmatic adversary who carefully selects targets and make predictions conservatively. We design a new evaluation methodology that allows us to evaluate the membership privacy risk at the level of individuals and not only in aggregate. We experimentally demonstrate that highly vulnerable records exist even when the aggregate attack precision is close to 50% (baseline). Specifically, on the MNIST dataset, our pragmatic adversary achieves a precision of 95.05% whereas the prior attack only achieves a precision of 51.7%.

MOTIVATION: Third generation sequencing techniques, such as the Single Molecule Real Time technique from PacBio and the MinION technique from Oxford Nanopore, can generate long, error-prone sequencing reads which pose new challenges for fragment assembly algorithms. In this paper, we study the overlap detection problem for error-prone reads, which is the first and most critical step in the de novo fragment assembly. We observe that all the state-of-the-art methods cannot achieve an ideal accuracy for overlap detection (in terms of relatively low precision and recall) due to the high sequencing error rates, especially when the overlap lengths between reads are relatively short (e.g. <2000 bases). This limitation appears inherent to these algorithms due to their usage of q-gram-based seeds under the seed-extension framework. RESULTS: We propose smooth q-gram, a variant of q-gram that captures q-gram pairs within small edit distances and design a novel algorithm for detecting overlapping reads using smooth q-gram-based seeds. We implemented the algorithm and tested it on both PacBio and Nanopore sequencing datasets. Our benchmarking results demonstrated that our algorithm outperforms the existing q-gram-based overlap detection algorithms, especially for reads with relatively short overlapping lengths. AVAILABILITY AND IMPLEMENTATION: The source code of our implementation in C++ is available at https://github.com/FIGOGO/smoothq. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: The generalized linear mixed model (GLMM) is an extension of the generalized linear model (GLM) in which the linear predictor takes random effects into account. Given its power of precisely modeling the mixed effects from multiple sources of random variations, the method has been widely used in biomedical computation, for instance in the genome-wide association studies (GWASs) that aim to detect genetic variance significantly associated with phenotypes such as human diseases. Collaborative GWAS on large cohorts of patients across multiple institutions is often impeded by the privacy concerns of sharing personal genomic and other health data. To address such concerns, we present in this paper a privacy-preserving Expectation-Maximization (EM) algorithm to build GLMM collaboratively when input data are distributed to multiple participating parties and cannot be transferred to a central server. We assume that the data are horizontally partitioned among participating parties: i.e. each party holds a subset of records (including observational values of fixed effect variables and their corresponding outcome), and for all records, the outcome is regulated by the same set of known fixed effects and random effects. RESULTS: Our collaborative EM algorithm is mathematically equivalent to the original EM algorithm commonly used in GLMM construction. The algorithm also runs efficiently when tested on simulated and real human genomic data, and thus can be practically used for privacy-preserving GLMM construction. We implemented the algorithm for collaborative GLMM (cGLMM) construction in R. The data communication was implemented using the rsocket package. AVAILABILITY AND IMPLEMENTATION: The software is released in open source at https://github.com/huthvincent/cGLMM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The four papers in this special section were presented at the 4th International Workshop on Genome Privacy and Security (GenoPri) in 2017. This workshop aimed to bring together a highly interdisciplinary community involved in all aspects of genome privacy and security research. This workshop built on its three predecessors, GenoPri’14, GenoPri’15, and GenoPri’16 which were collocated with the Privacy Enhancing Technologies Symposium (PETS), IEEE Symposium on Security and Privacy, and American Medical Informatics Association Annual Fall Symposium (AMIA), respectively. Over the past several decades, genome sequencing technologies have evolved from slow and expensive systems that were limited in access to a select few scientists and forensics investigators to high-throughput, relatively low-cost tools that are available to consumers.

We propose smooth q-gram, the first variant of q-gram that captures q-gram pair within a small edit distance. We apply smooth q-gram to the problem of detecting overlapping pairs of error-prone reads produced by single molecule real time sequencing (SMRT), which is the first and most critical step of the de novo fragment assembly of SMRT reads. We have implemented and tested our algorithm on a set of real world benchmarks. Our empirical results demonstrated the significant superiority of our algorithm over the existing q-gram based algorithms in accuracy.

Motivation: Rapid advancement in high throughput genome and transcriptome sequencing (HTS) and mass spectrometry (MS) technologies has enabled the acquisition of the genomic, transcriptomic and proteomic data from the same tissue sample. We introduce a computational framework, ProTIE, to integratively analyze all three types of omics data for a complete molecular profile of a tissue sample. Our framework features MiStrVar, a novel algorithmic method to identify micro structural variants (microSVs) on genomic HTS data. Coupled with deFuse, a popular gene fusion detection method we developed earlier, MiStrVar can accurately profile structurally aberrant transcripts in tumors. Given the breakpoints obtained by MiStrVar and deFuse, our framework can then identify all relevant peptides that span the breakpoint junctions and match them with unique proteomic signatures. Observing structural aberrations in all three types of omics data validates their presence in the tumor samples. Results: We have applied our framework to all The Cancer Genome Atlas (TCGA) breast cancer Whole Genome Sequencing (WGS) and/or RNA-Seq datasets, spanning all four major subtypes, for which proteomics data from Clinical Proteomic Tumor Analysis Consortium (CPTAC) have been released. A recent study on this dataset focusing on SNVs has reported many that lead to novel peptides. Complementing and significantly broadening this study, we detected 244 novel peptides from 432 candidate genomic or transcriptomic sequence aberrations. Many of the fusions and microSVs we discovered have not been reported in the literature. Interestingly, the vast majority of these translated aberrations, fusions in particular, were private, demonstrating the extensive inter-genomic heterogeneity present in breast cancer. Many of these aberrations also have matching out-of-frame downstream peptides, potentially indicating novel protein sequence and structure. Availability and implementation: MiStrVar is available for download at https://bitbucket.org/compbio/mistrvar, and ProTIE is available at https://bitbucket.org/compbio/protie. Contact: [email protected]. Supplementary information: Supplementary data are available at Bioinformatics online.

Side-channel risks of Intel's SGX have recently attracted great attention. Under the spotlight is the newly discovered page-fault attack, in which an OS-level adversary induces page faults to observe the page-level access patterns of a protected process running in an SGX enclave. With almost all proposed defense focusing on this attack, little is known about whether such efforts indeed raises the bar for the adversary, whether a simple variation of the attack renders all protection ineffective, not to mention an in-depth understanding of other attack surfaces in the SGX system. In the paper, we report the first step toward systematic analyses of side-channel threats that SGX faces, focusing on the risks associated with its memory management. Our research identifies 8 potential attack vectors, ranging from TLB to DRAM modules. More importantly, we highlight the common misunderstandings about SGX memory side channels, demonstrating that high frequent AEXs can be avoided when recovering EdDSA secret key through a new page channel and fine-grained monitoring of enclave programs (at the level of 64B) can be done through combining both cache and cross-enclave DRAM channels. Our findings reveal the gap between the ongoing security research on SGX and its side-channel weaknesses, redefine the side-channel threat model for secure enclaves, and can provoke a discussion on when to use such a system and how to use it securely.

MOTIVATION: The insertion sequence (IS) elements are the smallest but most abundant autonomous transposable elements in prokaryotic genomes, which play a key role in prokaryotic genome organization and evolution. With the fast growing genomic data, it is becoming increasingly critical for biology researchers to be able to accurately and automatically annotate ISs in prokaryotic genome sequences. The available automatic IS annotation systems are either providing only incomplete IS annotation or relying on the availability of existing genome annotations. Here, we present a new IS elements annotation pipeline to address these issues. RESULTS: ISEScan is a highly sensitive software pipeline based on profile hidden Markov models constructed from manually curated IS elements. ISEScan performs better than existing IS annotation systems when tested on prokaryotic genomes with curated annotations of IS elements. Applying it to 2784 prokaryotic genomes, we report the global distribution of IS families across taxonomic clades in Archaea and Bacteria. AVAILABILITY AND IMPLEMENTATION: ISEScan is implemented in Python and released as an open source software at https://github.com/xiezhq/ISEScan. CONTACT: [email protected]. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BACKGROUND: Short tandem repeats (STRs) are found in many prokaryotic and eukaryotic genomes, and are commonly used as genetic markers, in particular for identity and parental testing in DNA forensics. The unstable expansion of some STRs was associated with various genetic disorders (e.g., the Huntington disease), and thus was used in genetic testing for screening individuals at high risk. Traditional STR analyses were based on the PCR amplification of STR loci followed by gel electrophoresis. With the availability of massive whole genome sequencing data, it becomes practical to mine STR profiles in silico from genome sequences. Software tools such as lobSTR and STR-FM have been developed to address these demands, which are, however, built upon whole genome reads mapping tools, and thus may not be sensitive enough. RESULTS: In this paper, we present a standalone software tool STRScan that uses a greedy algorithm for targeted STR profiling in next-generation sequencing (NGS) data. STRScan was tested on the whole genome sequencing data from Venter genome sequencing and 1000 Genomes Project. The results showed that STRScan can profile 20% more STRs in the target set that are missed by lobSTR. CONCLUSION: STRScan is particularly useful for the NGS-based targeted STR profiling, e.g., in genetic and human identity testing. STRScan is available as open-source software at http://darwin.informatics.indiana.edu/str/ .

The Global Alliance for Genomics and Health (GA4GH) created the Beacon Project as a means of testing the willingness of data holders to share genetic data in the simplest technical context-a query for the presence of a specified nucleotide at a given position within a chromosome. Each participating site (or "beacon") is responsible for assuring that genomic data are exposed through the Beacon service only with the permission of the individual to whom the data pertains and in accordance with the GA4GH policy and standards.While recognizing the inference risks associated with large-scale data aggregation, and the fact that some beacons contain sensitive phenotypic associations that increase privacy risk, the GA4GH adjudged the risk of re-identification based on the binary yes/no allele-presence query responses as acceptable. However, recent work demonstrated that, given a beacon with specific characteristics (including relatively small sample size and an adversary who possesses an individual's whole genome sequence), the individual's membership in a beacon can be inferred through repeated queries for variants present in the individual's genome.In this paper, we propose three practical strategies for reducing re-identification risks in beacons. The first two strategies manipulate the beacon such that the presence of rare alleles is obscured; the third strategy budgets the number of accesses per user for each individual genome. Using a beacon containing data from the 1000 Genomes Project, we demonstrate that the proposed strategies can effectively reduce re-identification risk in beacon-like datasets.

UNLABELLED: : MGEScan-long terminal repeat (LTR) and MGEScan-non-LTR are successfully used programs for identifying LTRs and non-LTR retrotransposons in eukaryotic genome sequences. However, these programs are not supported by easy-to-use interfaces nor well suited for data visualization in general data formats. Here, we present MGEScan, a user-friendly system that combines these two programs with a Galaxy workflow system accelerated with MPI and Python threading on compute clusters. MGEScan and Galaxy empower researchers to identify transposable elements in a graphical user interface with ready-to-use workflows. MGEScan also visualizes the custom annotation tracks for mobile genetic elements in public genome browsers. A maximum speed-up of 3.26× is attained for execution time using concurrent processing and MPI on four virtual cores. MGEScan provides four operational modes: as a command line tool, as a Galaxy Toolshed, on a Galaxy-based web server, and on a virtual cluster on the Amazon cloud. AVAILABILITY AND IMPLEMENTATION: MGEScan tutorials and source code are available at http://mgescan.readthedocs.org/ CONTACT: [email protected] or [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: Metagenomics research has accelerated the studies of microbial organisms, providing insights into the composition and potential functionality of various microbial communities. Metatranscriptomics (studies of the transcripts from a mixture of microbial species) and other meta-omics approaches hold even greater promise for providing additional insights into functional and regulatory characteristics of the microbial communities. Current metatranscriptomics projects are often carried out without matched metagenomic datasets (of the same microbial communities). For the projects that produce both metatranscriptomic and metagenomic datasets, their analyses are often not integrated. Metagenome assemblies are far from perfect, partially explaining why metagenome assemblies are not used for the analysis of metatranscriptomic datasets. RESULTS: Here, we report a reads mapping algorithm for mapping of short reads onto a de Bruijn graph of assemblies. A hash table of junction k-mers (k-mers spanning branching structures in the de Bruijn graph) is used to facilitate fast mapping of reads to the graph. We developed an application of this mapping algorithm: a reference-based approach to metatranscriptome assembly using graphs of metagenome assembly as the reference. Our results show that this new approach (called TAG) helps to assemble substantially more transcripts that otherwise would have been missed or truncated because of the fragmented nature of the reference metagenome. AVAILABILITY AND IMPLEMENTATION: TAG was implemented in C++ and has been tested extensively on the Linux platform. It is available for download as open source at http://omics.informatics.indiana.edu/TAG CONTACT: [email protected].

Metaproteomic studies adopt the common bottom-up proteomics approach to investigate the protein composition and the dynamics of protein expression in microbial communities. When matched metagenomic and/or metatranscriptomic data of the microbial communities are available, metaproteomic data analyses often employ a metagenome-guided approach, in which complete or fragmental protein-coding genes are first directly predicted from metagenomic (and/or metatranscriptomic) sequences or from their assemblies, and the resulting protein sequences are then used as the reference database for peptide/protein identification from MS/MS spectra. This approach is often limited because protein coding genes predicted from metagenomes are incomplete and fragmental. In this paper, we present a graph-centric approach to improving metagenome-guided peptide and protein identification in metaproteomics. Our method exploits the de Bruijn graph structure reported by metagenome assembly algorithms to generate a comprehensive database of protein sequences encoded in the community. We tested our method using several public metaproteomic datasets with matched metagenomic and metatranscriptomic sequencing data acquired from complex microbial communities in a biological wastewater treatment plant. The results showed that many more peptides and proteins can be identified when assembly graphs were utilized, improving the characterization of the proteins expressed in the microbial communities. The additional proteins we identified contribute to the characterization of important pathways such as those involved in degradation of chemical hazards. Our tools are released as open-source software on github at https://github.com/COL-IU/Graph2Pro.

Edit distance has been proven to be an important and frequently-used metric in many human genomic research, with Similar Patient Query (SPQ) being a particularly promising and attractive example. However, due to the widespread privacy concerns on revealing personal genomic data, the scope and scale of many novel use of genome edit distance are substantially limited. While the problem of private genomic edit distance has been studied by the research community for over a decade [6], the state-of-the-art solution [31] is far from even close to be applicable to real genome sequences. In this paper, we propose several private edit distance protocols that feature unprecedentedly high efficiency and precision. Our construction is a combination of a novel genomic edit distance ap- proximation algorithm and new construction of private set difference size protocols. With the private edit distance based secure SPQ primitive, we propose GENSETS, a genome-wide, privacy- preserving similar patient query system. It is able to support search- ing large-scale, distributed genome databases across the nation. We have implemented a prototype of GENSETS. The experimental results show that, with 100 Mbps network connection, it would take GENSETS less than 200 minutes to search through 1 million breast cancer patients (distributed nation-wide in 250 hospitals, each having 4000 patients), based on edit distances between their genomes of lengths about 75 million nucleotides each.

OBJECTIVE: To propose a new approach to privacy preserving data selection, which helps the data users access human genomic datasets efficiently without undermining patients' privacy. METHODS: Our idea is to let each data owner publish a set of differentially-private pilot data, on which a data user can test-run arbitrary association-test algorithms, including those not known to the data owner a priori. We developed a suite of new techniques, including a pilot-data generation approach that leverages the linkage disequilibrium in the human genome to preserve both the utility of the data and the privacy of the patients, and a utility evaluation method that helps the user assess the value of the real data from its pilot version with high confidence. RESULTS: We evaluated our approach on real human genomic data using four popular association tests. Our study shows that the proposed approach can help data users make the right choices in most cases. CONCLUSIONS: Even though the pilot data cannot be directly used for scientific discovery, it provides a useful indication of which datasets are more likely to be useful to data users, who can therefore approach the appropriate data owners to gain access to the data.

The viral quasispecies represent a set of related variants in a virus population (e.g. from an infected patient) that contain similar mutations due to the rapid and mutation-prone replications in viruses. The characterization of viral quasispecies in a highly divergent virus population is of great interest in biomedical research, in particular, to identify virulent and drug-resistant mutations in viral genomes for diagnosis of infectious diseases and targeted drug design. In recent years, next-generation sequencing (NGS) techniques have been widely used for deep sequencing of virus populations, in an attempt to characterize low abundant viral quasispecies containing specific mutations associated with virulence or drug-resistance. However, because of the short length of NGS reads, it remains a challenge to reconstruct viral quasispecies from NGS sequencing data. In this paper, we formulate the viral quasispecies reconstruction as the vertex coloring problem on a read conflict graph, and then apply heuristic algorithms to solve it. We compared our new algorithms with one existing software tool on three simulated datasets for HIV quasispecies reconstruction. The results showed our methods can improve the accuracy on the inference of the identities and quantities of viral quansispecies in a virus population.

Accessory genomes in bacterial species carry important genetic elements that are frequently related to antibiotic resistance, virulence factors, and the biotransformation of xenobiotics. Facilitated by the recent advances in sequencing technology, bacterial genomes and metagenomes are accumulating at an unprecedented pace, providing opportunities for studies of accessory genomes. Comparison of closely related genomes reveals potential (and static) accessory genomes, and metagenomic recruitment (i.e., mapping metagenomic sequences onto reference genomes) provides insights into the nature and the dynamics of the accessory portion of the genomes. Recent metagenomic recruitment approaches focus on the identification of `metagenomic islands' (MIs), segments in reference genomes that are under-recruiting in metagenomic samples and therefore likely to be mobile genetic elements (MGEs) in accessory genomes. However, the discovery of MIs often relies on manual inspection of the read recruitment plots. Here we introduce a method that integrates comparison of closely related genomes using A-Bruijn graph, metagenomic recruitment, and recurrent analysis for the identification and characterization of accessory genomes. In addition to metage-nomic islands (valleys), our method reveals `metagenomic peaks' (MPs), segments in a reference genome that disproportionally recruit more metagenomic sequencing reads as compared to the remaining of the reference genome, indicating an enrichment of those segments in specific environments. Our method facilitates automated detection and characterization of accessory genomes at a large scale, and leads to the observation that MGEs are largely specific to environments, as demonstrated in the discovery of MGEs related to Streptococcus mitis in human microbiomes.

Phylogenetic analysis is commonly used to analyze genetic sequence data from fungal communities, while ordination and clustering techniques commonly are used to analyze sequence data from bacterial communities. However, few studies have attempted to link these two independent approaches. In this paper, we propose a method, which we call spherical phylogram (SP), to display the phylogenetic tree within the clustering and visualization result from a pipeline called DACIDR. In comparison with traditional tree display methods, the correlations between the tree and the clustering can be observed directly. In addition, we propose an algorithm called interpolative joining (IJ) to construct and visualize the SP in 3D space. In the experiments, we used the sum of branch lengths to quantify the general fit between the clustering and the phylogenetic tree in SP and Mantel tests to determine how well the same grouping of sequences was preserved between the clustering and the SP. Our results show that DACIDR has a classification accuracy that is similar to a phylogenetic tree generated using a multiple sequence alignment, while having much lower computational cost.

BACKGROUND: Metatranscriptomic sequencing is a highly sensitive bioassay of functional activity in a microbial community, providing complementary information to the metagenomic sequencing of the community. The acquisition of the metatranscriptomic sequences will enable us to refine the annotations of the metagenomes, and to study the gene activities and their regulation in complex microbial communities and their dynamics. RESULTS: In this paper, we present TransGeneScan, a software tool for finding genes in assembled transcripts from metatranscriptomic sequences. By incorporating several features of metatranscriptomic sequencing, including strand-specificity, short intergenic regions, and putative antisense transcripts into a Hidden Markov Model, TranGeneScan can predict a sense transcript containing one or multiple genes (in an operon) or an antisense transcript. CONCLUSION: We tested TransGeneScan on a mock metatranscriptomic data set containing three known bacterial genomes. The results showed that TranGeneScan performs better than metagenomic gene finders (MetaGeneMark and FragGeneScan) on predicting protein coding genes in assembled transcripts, and achieves comparable or even higher accuracy than gene finders for microbial genomes (Glimmer and GeneMark). These results imply, with the assistance of metatranscriptomic sequencing, we can obtain a broad and precise picture about the genes (and their functions) in a microbial community. AVAILABILITY: TransGeneScan is available as open-source software on SourceForge at https://sourceforge.net/projects/transgenescan/.

UNLABELLED: As a common post-translational modification, protein glycosylation plays an important role in many biological processes, and it is known to be associated with human diseases. Mass spectrometry (MS)-based glycomic profiling techniques have been developed to measure the abundances of glycans in complex biological samples and applied to the discovery of putative glycan biomarkers. To automate the annotation of glycomic profiles in the liquid chromatography-MS (LC-MS) data, we present here a user-friendly software tool, MultiGlycan, implemented in C# on Windows systems. We tested MultiGlycan by using several glycomic profiling datasets acquired using LC-MS under different preparations and show that MultiGlycan executes fast and generates robust and reliable results. AVAILABILITY: MultiGlycan can be freely downloaded at http://darwin.informatics.indiana.edu/MultiGlycan/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Shotgun metagenomics has been applied to the studies of the functionality of various microbial communities. As a critical analysis step in these studies, biological pathways are reconstructed based on the genes predicted from metagenomic shotgun sequences. Pathway reconstruction provides insights into the functionality of a microbial community and can be used for comparing multiple microbial communities. The utilization of pathway reconstruction, however, can be jeopardized because of imperfect functional annotation of genes, and ambiguity in the assignment of predicted enzymes to biochemical reactions (e.g., some enzymes are involved in multiple biochemical reactions). Considering that metabolic functions in a microbial community are carried out by many enzymes in a collaborative manner, we present a probabilistic sampling approach to profiling functional content in a metagenomic dataset, by sampling functions of catalytically promiscuous enzymes within the context of the entire metabolic network defined by the annotated metagenome. We test our approach on metagenomic datasets from environmental and human-associated microbial communities. The results show that our approach provides a more accurate representation of the metabolic activities encoded in a metagenome, and thus improves the comparative analysis of multiple microbial communities. In addition, our approach reports likelihood scores of putative reactions, which can be used to identify important reactions and metabolic pathways that reflect the environmental adaptation of the microbial communities. Source code for sampling metabolic networks is available online at http://omics.informatics.indiana.edu/mg/MetaNetSam/.

SUMMARY: With the wide application of next-generation sequencing (NGS) techniques, fast tools for protein similarity search that scale well to large query datasets and large databases are highly desirable. In a previous work, we developed RAPSearch, an algorithm that achieved a ~20-90-fold speedup relative to BLAST while still achieving similar levels of sensitivity for short protein fragments derived from NGS data. RAPSearch, however, requires a substantial memory footprint to identify alignment seeds, due to its use of a suffix array data structure. Here we present RAPSearch2, a new memory-efficient implementation of the RAPSearch algorithm that uses a collision-free hash table to index a similarity search database. The utilization of an optimized data structure further speeds up the similarity search-another 2-3 times. We also implemented multi-threading in RAPSearch2, and the multi-thread modes achieve significant acceleration (e.g. 3.5X for 4-thread mode). RAPSearch2 requires up to 2G memory when running in single thread mode, or up to 3.5G memory when running in 4-thread mode. AVAILABILITY AND IMPLEMENTATION: Implemented in C++, the source code is freely available for download at the RAPSearch2 website: http://omics.informatics.indiana.edu/mg/RAPSearch2/. CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Available at the RAPSearch2 website.

Discordant read pairs [ 1 , 2 ] – those deviating either from expected insert size range or correct relative orientation – have served as vital clues to identifying structural variants (SV) in genomes. Collecting discordant read pairs is the first step in SV detection and is often done by sequence alignment. When there are repetitive elements, such as insertion sequence (IS), a class of transposable elements in bacterial genomes, discordant read pairs can have multiple mapping loci – making them more challenging to be placed and interpreted. Instead of resolving such tangled mapping results, many tools simply ignore these mapped read pairs, potentially missing SVs involving repetitive elements. We present an idea of using approximate de Bruijn graphs ( A -Bruijn graphs) [ 3 ] to identify discordant read pairs, in order to discover SVs. Repeats are easily recognized in A -Bruijn graphs, as all repetitive elements of the same kind are collapsed into a contiguous edge. When read pairs representing repetitive elements are mapped to a reference A -Bruijn graph, only those from novel insertions are flagged as discordant and the rest – those from preexisting insertion loci – mapped concordantly. We applied this approach to whole genome sequencing data [ 4 ] (~100x per sample using 90bp x 2 paired end Illumina sequencing) obtained from 38 lines of Escherichia coli PFM2, a derivative strain of E. coli K-12 MG1655, and 34 lines of a mismatch repair deficient (deletion of mutL ) derivative that were propagated for ~3,080 and ~375 generations respectively via a mutation accumulation (MA) strategy. All of the inferred IS insertions were directly confirmed by PCR experiments. A total of 27 IS transpositions has been detected and includes 5 out of 12 IS families present in E. coli K-12. We have also identified an insertion of IS186 that is fixed among all MA lines and not present in the reference E. coli genome. 24 out 27 inferred insertions were validated by PCR and 3 of them are currently under analysis. The fixed insertion of IS186 in the samples was also confirmed by PCR. Our method can pinpoint SVs by identifying discordant read pairs resulting from novel insertions of repetitive elements, where many other currently available tools fail. This result serves as a first step towards inferring the neutral rate of IS transposition in bacterial genomes.

BACKGROUND: Quantification of protein expression by means of mass spectrometry (MS) has been introduced in various proteomics studies. In particular, two label-free quantification methods, such as spectral counting and spectra feature analysis have been extensively investigated in a wide variety of proteomic studies. The cornerstone of both methods is peptide identification based on a proteomic database search and subsequent estimation of peptide retention time. However, they often suffer from restrictive database search and inaccurate estimation of the liquid chromatography (LC) retention time. Furthermore, conventional peptide identification methods based on the spectral library search algorithms such as SEQUEST or SpectraST have been found to provide neither the best match nor high-scored matches. Lastly, these methods are limited in the sense that target peptides cannot be identified unless they have been previously generated and stored into the database or spectral libraries.To overcome these limitations, we propose a novel method, namely Quantification method based on Finding the Identical Spectral set for a Homogenous peptide (Q-FISH) to estimate the peptide's abundance from its tandem mass spectrometry (MS/MS) spectra through the direct comparison of experimental spectra. Intuitively, our Q-FISH method compares all possible pairs of experimental spectra in order to identify both known and novel proteins, significantly enhancing identification accuracy by grouping replicated spectra from the same peptide targets. RESULTS: We applied Q-FISH to Nano-LC-MS/MS data obtained from human hepatocellular carcinoma (HCC) and normal liver tissue samples to identify differentially expressed peptides between the normal and disease samples. For a total of 44,318 spectra obtained through MS/MS analysis, Q-FISH yielded 14,747 clusters. Among these, 5,777 clusters were identified only in the HCC sample, 6,648 clusters only in the normal tissue sample, and 2,323 clusters both in the HCC and normal tissue samples. While it will be interesting to investigate peptide clusters only found from one sample, further examined spectral clusters identified both in the HCC and normal samples since our goal is to identify and assess differentially expressed peptides quantitatively. The next step was to perform a beta-binomial test to isolate differentially expressed peptides between the HCC and normal tissue samples. This test resulted in 84 peptides with significantly differential spectral counts between the HCC and normal tissue samples. We independently identified 50 and 95 peptides by SEQUEST, of which 24 and 56 peptides, respectively, were found to be known biomarkers for the human liver cancer. Comparing Q-FISH and SEQUEST results, we found 22 of the differentially expressed 84 peptides by Q-FISH were also identified by SEQUEST. Remarkably, of these 22 peptides discovered both by Q-FISH and SEQUEST, 13 peptides are known for human liver cancer and the remaining 9 peptides are known to be associated with other cancers. CONCLUSIONS: We proposed a novel statistical method, Q-FISH, for accurately identifying protein species and simultaneously quantifying the expression levels of identified peptides from mass spectrometry data. Q-FISH analysis on human HCC and liver tissue samples identified many protein biomarkers that are highly relevant to HCC. Q-FISH can be a useful tool both for peptide identification and quantification on mass spectrometry data analysis. It may also prove to be more effective in discovering novel protein biomarkers than SEQUEST and other standard methods.

BACKGROUND: Next Generation Sequencing (NGS) is producing enormous corpuses of short DNA reads, affecting emerging fields like metagenomics. Protein similarity search--a key step to achieve annotation of protein-coding genes in these short reads, and identification of their biological functions--faces daunting challenges because of the very sizes of the short read datasets. RESULTS: We developed a fast protein similarity search tool RAPSearch that utilizes a reduced amino acid alphabet and suffix array to detect seeds of flexible length. For short reads (translated in 6 frames) we tested, RAPSearch achieved ~20-90 times speedup as compared to BLASTX. RAPSearch missed only a small fraction (~1.3-3.2%) of BLASTX similarity hits, but it also discovered additional homologous proteins (~0.3-2.1%) that BLASTX missed. By contrast, BLAT, a tool that is even slightly faster than RAPSearch, had significant loss of sensitivity as compared to RAPSearch and BLAST. CONCLUSIONS: RAPSearch is implemented as open-source software and is accessible at http://omics.informatics.indiana.edu/mg/RAPSearch. It enables faster protein similarity search. The application of RAPSearch in metageomics has also been demonstrated.

Genome-wide association studies (GWAS) aim at discovering the association between genetic variations, particularly single-nucleotide polymorphism (SNP), and common diseases, which is well recognized to be one of the most important and active areas in biomedical research. Also renowned is the privacy implication of such studies, which has been brought into the limelight by the recent attack proposed by Homer et al. Homer's attack demonstrates that it is possible to identify a GWAS participant from the allele frequencies of a large number of SNPs. Such a threat, unfortunately, was found in our research to be significantly understated. In this paper, we show that individuals can actually be identified from even a relatively small set of statistics, as those routinely published in GWAS papers. We present two attacks. The first one extends Homer's attack with a much more powerful test statistic, based on the correlations among different SNPs described by coefficient of determination (r2). This attack can determine the presence of an individual from the statistics related to a couple of hundred SNPs. The second attack can lead to complete disclosure of hundreds of participants' SNPs, through analyzing the information derived from published statistics. We also found that those attacks can succeed even when the precisions of the statistics are low and part of data is missing. We evaluated our attacks on the real human genomes and concluded that such threats are completely realistic.

In this paper, we present a new approach to performing important classes of genomic computations (e.g., search for homologous genes) that makes a significant step towards privacy protection in this domain. Our approach leverages a key property of the human genome, namely that the vast majority of it is shared across humans (and hence public), and consequently relatively little of it is sensitive. Based on this observation, we propose a privacy-protection framework that partitions a genomic computation, distributing the part on sensitive data to the data provider and the part on the pubic data to the user of the data. Such a partition is achieved through program specialization that enables a biocomputing program to perform a concrete execution on public data and a symbolic execution on sensitive data. As a result, the program is simplified into an efficient query program that takes only sensitive genetic data as inputs. We prove the effectiveness of our techniques on a set of dynamic programming algorithms fundamental to genomic computing. We develop a program transformation tool that automatically instruments a legacy program for specialization operations. We also demonstrate that our techniques can greatly facilitate secure multi-party computations on large biocomputing problems.

MOTIVATION: One of the major problems in shotgun proteomics is the low peptide coverage when analyzing complex protein samples. Identifying more peptides, e.g. non-tryptic peptides, may increase the peptide coverage and improve protein identification and/or quantification that are based on the peptide identification results. Searching for all potential non-tryptic peptides is, however, time consuming for shotgun proteomics data from complex samples, and poses a challenge for a routine data analysis. RESULTS: We hypothesize that non-tryptic peptides are mainly created from the truncation of regular tryptic peptides before separation. We introduce the notion of truncatability of a tryptic peptide, i.e. the probability of the peptide to be identified in its truncated form, and build a predictor to estimate a peptide's truncatability from its sequence. We show that our predictions achieve useful accuracy, with the area under the ROC curve from 76% to 87%, and can be used to filter the sequence database for identifying truncated peptides. After filtering, only a limited number of tryptic peptides with the highest truncatability are retained for non-tryptic peptide searching. By applying this method to identification of semi-tryptic peptides, we show that a significant number of such peptides can be identified within a searching time comparable to that of tryptic peptide identification.

MOTIVATION: While it is common to refer to 'the genome sequence' as if it were a single, complete and contiguous DNA string, it is in fact an assembly of millions of small, partially overlapping DNA fragments. Sophisticated computer algorithms (assemblers and scaffolders) merge these DNA fragments into contigs, and place these contigs into sequence scaffolds using the paired-end sequences derived from large-insert DNA libraries. Each step in this automated process is susceptible to producing errors; hence, the resulting draft assembly represents (in practice) only a likely assembly that requires further validation. Knowing which parts of the draft assembly are likely free of errors is critical if researchers are to draw reliable conclusions from the assembled sequence data. RESULTS: We develop a machine-learning method to detect assembly errors in sequence assemblies. Several in silico measures for assembly validation have been proposed by various researchers. Using three benchmarking Drosophila draft genomes, we evaluate these techniques along with some new measures that we propose, including the good-minus-bad coverage (GMB), the good-to-bad-ratio (RGB), the average Z-score (AZ) and the average absolute Z-score (ASZ). Our results show that the GMB measure performs better than the others in both its sensitivity and its specificity for assembly error detection. Nevertheless, no single method performs sufficiently well to reliably detect genomic regions requiring attention for further experimental verification. To utilize the advantages of all these measures, we develop a novel machine learning approach that combines these individual measures to achieve a higher prediction accuracy (i.e. greater than 90%). Our combined evidence approach avoids the difficult and often ad hoc selection of many parameters the individual measures require, and significantly improves the overall precisions on the benchmarking data sets.

Abstract Motivation: Transcription factor binding sites (TFBSs) are typically short in length, thus search with a profile model from known TFBSs produces many false positives. When combined with additional information, gene expression data in this article, sensitivity and specificity of TFBS search can be improved significantly. Results: By modifying our previous REFINEMENT approach, we developed dPattern that searches for occurrences of TFBSs in the promotor regions of up/down regulated or random genes. Availability: http://platcom.org/projects/dpattern Contact: [email protected] or [email protected]

Accurate base-assignment in repeat regions of a whole genome shotgun assembly is an unsolved problem. Since reads in repeat regions cannot be easily attributed to a unique location in the genome, current assemblers may place these reads arbitrarily. As a result, the base-assignment error rate in repeats is likely to be much higher than that in the rest of the genome. We developed an iterative algorithm, EULER-AIR, that is able to correct base-assignment errors in finished genome sequences in public databases. The Wolbachia genome is among the best finished genomes. Using this genome project as an example, we demonstrated that EULER-AIR can 1) discover and correct base-assignment errors, 2) provide accurate read assignments, 3) utilize finishing reads for accurate base-assignment, and 4) provide guidance for designing finishing experiments. In the genome of Wolbachia, EULER-AIR found 16 positions with ambiguous base-assignment and two positions with erroneous bases. Besides Wolbachia, many other genome sequencing projects have significantly fewer finishing reads and, hence, are likely to contain more base-assignment errors in repeats. We demonstrate that EULER-AIR is a software tool that can be used to find and correct base-assignment errors in a genome assembly project.

Repetitive sequences make up a significant fraction of almost any genome and an important and still open question in bioinformatics is how to represent all repeats in DNA sequences. We propose a radically new approach to repeat classification that is motivated by the fundamental topological notion of quotient spaces. A torus or Klein bottle are examples of quotient spaces that can be obtained from a square by gluing some points. Our new repeat classification algorithm is based on the observation that the alignment-induced quotient space of a DNA sequence compactly represents all sequence repeats. This observation leads to a simple and efficient solution of the repeat classification problem as well as new approaches to fragment assembly and multiple alignment.

MOTIVATION: Current DNA sequencing technology produces reads of about 500-750 bp, with typical coverage under 10x. New sequencing technologies are emerging that produce shorter reads (length 80-200 bp) but allow one to generate significantly higher coverage (30x and higher) at low cost. Modern assembly programs and error correction routines have been tuned to work well with current read technology but were not designed for assembly of short reads. RESULTS: We analyze the limitations of assembling reads generated by these new technologies and present a routine for base-calling in reads prior to their assembly. We demonstrate that while it is feasible to assemble such short reads, the resulting contigs will require significant (if not prohibitive) finishing efforts. AVAILABILITY: Available from the web at http://www.cse.ucsd.edu/groups/bioinformatics/software.html

UNLABELLED: In the genomic era, researchers often want to know more information about a biological sequence by retrieving its related articles. However, there is no available tool yet to achieve conveniently this goal. Here we developed a new literature-mining tool MedBlast, which uses natural language processing techniques, to retrieve the related articles of a given sequence. An online server of this program is also provided. AVAILABILITY: Both online server and the program are available freely at http://medblast.sibsnet.org

MOTIVATION: The traditional approach to annotate alternative splicing is to investigate every splicing variant of the gene in a case-by-case fashion. This approach, while useful, has some serious shortcomings. Recent studies indicate that alternative splicing is more frequent than previously thought and some genes may produce tens of thousands of different transcripts. A list of alternatively spliced variants for such genes would be difficult to build and hard to analyse. Moreover, such a list does not show the relationships between different transcripts and does not show the overall structure of all transcripts. A better approach would be to represent all splicing variants for a given gene in a way that captures the relationships between different splicing variants. RESULTS: We introduce the notion of the splicing graph that is a natural and convenient representation of all splicing variants. The key difference with the existing approaches is that we abandon the linear (sequence) representation of each transcript and replace it with a graph representation where each transcript corresponds to a path in the graph. We further design an algorithm to assemble EST reads into the splicing graph rather than assembling them into each splicing variant in a case-by-case fashion.

For the last twenty years fragment assembly in DNA sequencing followed the “overlap - layout - consensus” paradigm that is used in all currently available assembly tools. Although this approach proved to be useful in assembling clones, it faces difficulties in genomic shotgun assembly: the existing algorithms make assembly errors and are often unable to resolve repeats even in prokaryotic genomes. Biologists are well-aware of these errors and are forced to carry additional experiments to verify the assembled contigs.