Abstract Motivation The Oxford Nanopore Technologies’ sequencing platform offers a path towards bedside genomics, producing long reads that can completely cover a gene of interest, and detect any known or novel variant the gene contains. However, the analysis of these long reads to identify actionable genotypes remains challenging and typically requires customization depending on the target gene. Results Here, we describe a generic algorithm to accurately reconstruct allele sequences derived from long-reads of amplicon-based data. Rather than calling variants directly from these long-reads, our method takes a “sequence-first” approach, performing an unbiased reconstruction of the underlying amplicon sequences to generate high-confidence reconstructed allele sequences. This is done without user input of the target gene, allowing for any source amplicon to be reconstructed. These high-confidence reconstructed allele sequences are then compared to the genomic reference sequence of the gene to infer the specific diplotype present in the sample. This approach is agnostic towards the number of genes and alleles present and readily detects novel variants. We demonstrate our approach using three independent data sets for CYP2D6, a diverse and complex gene with over 175 known alleles of clinical significance. We show how our approach can accurately recover validated CYP2D6 diplotypes from 20 Coriell samples covering 14 distinct alleles, using different amplicons, flow cell versions, and depths. This includes inferring occurrences of allele duplication events from relative abundances of each allele, a critical factor for ascribing functional effects to a diplotype. Further, we demonstrate our approach’s utility for other genomic regions, including HLA. Availability Custom code is available at the following GitHub repository, along with instructions for use and test data: https://github.com/scottdbrown/allele-reconstruction-long-read-amplicon-data. A snapshot of the code at the time of publication is available on Zenodo.org; doi 10.5281/zenodo.19716004. Raw .fastq sequence data for our three sequencing runs is available at the SRA under Bioproject PRJNA1357883 (https://www.ncbi.nlm.nih.gov/bioproject/1357883).

BACKGROUND: Sequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications. RESULTS: Here, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies' MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 to 0.53%, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100% accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing. CONCLUSIONS: Our pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required.

The introduction of next-generation DNA sequencing devices into the field of oncology provides an unprecedented mechanism to determine the underlying genetic changes that have occurred within a tumor and also the changes that accrue during treatment. An enhanced understanding of the oncogenic mechanisms could have an immediate clinical role in the treatment of rare tumors - where treatment protocols do not exist and their rarity would indicate that clinical trials would be unlikely to be undertaken for their establishment. We have investigated the utility of massively parallel sequencing to characterize a rare adenocarcinoma of the tongue, before and after treatment. In the pre-treatment tumor we identified 7,629 genes within regions of copy number gain, 1,078 genes exhibited increased expression relative to the blood and unrelated tumors and four genes contained somatic protein-coding mutations. Our analysis suggested the tumor cells were driven by the RET oncogene and its other pathway constituents. Genes whose protein products are targeted by the RET inhibitors sunitinib and sorafenib correlated with being amplified and or highly expressed. Consistent with our observations subsequent administration of sunitinib was associated with stable disease lasting 4 months, after which the lung lesions began to grow. Administration of sorafenib and sulindac provided disease stabilization for an additional 3 months after which the cancer progressed and new lesions appeared. A metastasis recurring in the skin was determined to possess 7,288 genes within copy number amplicons, 385 genes exhibiting increased expression relative to other tumours and 9 new somatic protein coding mutations. The observed mutations and amplifications were found to be consistent with resistance to therapy arising through further activation of RET pathway and nascent activation of the AKT pathway. Our results provide evidence for the clinical utility of complete genomic characterization and direct in-vivo genome-wide characterization of the mutations accruing within a tumor under drug selection.

MOTIVATION: T-cell receptor (TCR) diversity in peripheral blood has not yet been fully profiled with sequence level resolution. Each T-cell clonotype expresses a unique receptor, generated by somatic recombination of TCR genes and the enormous potential for T-cell diversity makes repertoire analysis challenging. We developed a sequencing approach and assembly software (immuno-SSAKE or iSSAKE) for profiling T-cell metagenomes using short reads from the massively parallel sequencing platforms. RESULTS: Models of sequence diversity for the TCR beta-chain CDR3 region were built using empirical data and used to simulate, at random, distinct TCR clonotypes at 1-20 p.p.m. Using simulated TCRbeta (sTCRbeta) sequences, we randomly created 20 million 36 nt reads having 1-2% random error, 20 million 42 or 50 nt reads having 1% random error and 20 million 36 nt reads with 1% error modeled on real short read data. Reads aligning to the end of known TCR variable (V) genes and having consecutive unmatched bases in the adjacent CDR3 were used to seed iSSAKE de novo assemblies of CDR3. With assembled 36 nt reads, we detect over 51% and 63% of rare (1 p.p.m.) clonotypes using a random or modeled error distribution, respectively. We detect over 99% of more abundant clonotypes (6 p.p.m. or higher) using either error distribution. Longer reads improve sensitivity, with assembled 42 and 50 nt reads identifying 82.0% and 94.7% of rare 1 p.p.m. clonotypes, respectively. Our approach illustrates the feasibility of complete profiling of the TCR repertoire using new massively parallel short read sequencing technology. AVAILABILITY: ftp://ftp.bcgsc.ca/supplementary/iSSAKE.

UNLABELLED: Novel DNA sequencing technologies with the potential for up to three orders magnitude more sequence throughput than conventional Sanger sequencing are emerging. The instrument now available from Solexa Ltd, produces millions of short DNA sequences of 25 nt each. Due to ubiquitous repeats in large genomes and the inability of short sequences to uniquely and unambiguously characterize them, the short read length limits applicability for de novo sequencing. However, given the sequencing depth and the throughput of this instrument, stringent assembly of highly identical sequences can be achieved. We describe SSAKE, a tool for aggressively assembling millions of short nucleotide sequences by progressively searching through a prefix tree for the longest possible overlap between any two sequences. SSAKE is designed to help leverage the information from short sequence reads by stringently assembling them into contiguous sequences that can be used to characterize novel sequencing targets. AVAILABILITY: http://www.bcgsc.ca/bioinfo/software/ssake.

BACKGROUND: To date, 35 human diseases, some of which also exhibit anticipation, have been associated with unstable repeats. Anticipation has been reported in a number of diseases in which repeat expansion may have a role in etiology. Despite the growing importance of unstable repeats in disease, currently no resource exists for the prioritization of repeats. Here we present Satellog, a database that catalogs all pure 1-16 repeat unit satellite repeats in the human genome along with supplementary data. Satellog analyzes each pure repeat in UniGene clusters for evidence of repeat polymorphism. RESULTS: A total of 5,546 such repeats were identified, providing the first indication of many novel polymorphic sites in the genome. Overall, polymorphic repeats were over-represented within 3'-UTR sequence relative to 5'-UTR and coding sequence. Interestingly, we observed that repeat polymorphism within coding sequence is restricted to trinucleotide repeats whereas UTR sequence tolerated a wider range of repeat period polymorphisms. For each pure repeat we also calculate its repeat length percentile rank, its location either within or adjacent to EnsEMBL genes, and its expression profile in normal tissues according to the GeneNote database. CONCLUSION: Satellog provides the ability to dynamically prioritize repeats based on any of their characteristics (i.e. repeat unit, class, period, length, repeat length percentile rank, genomic co-ordinates), polymorphism profile within UniGene, proximity to or presence within gene regions (i.e. cds, UTR, 15 kb upstream etc.), metadata of the genes they are detected within and gene expression profiles within normal human tissues. Unstable repeats associated with 31 diseases were analyzed in Satellog to evaluate their common repeat properties. The utility of Satellog was highlighted by prioritizing repeats for Huntington's disease and schizophrenia. Satellog is available online at http://satellog.bcgsc.ca.