Motivation: Targeted sequencing using capture probes has become increasingly popular in clinical applications due to its scalability and cost-effectiveness. The approach also allows for higher sequencing coverage of the targeted regions resulting in better analysis statistical power. However, because of the dynamics of the hybridization process, it is difficult to evaluate the efficiency of the probe design prior to the experiments which are time consuming and costly. Results: We developed CapSim, a software package for simulation of targeted sequencing. Given a genome sequence and a set of probes, CapSim simulates the fragmentation, the dynamics of probe hybridization and the sequencing of the captured fragments on Illumina and PacBio sequencing platforms. The simulated data can be used for evaluating the performance of the analysis pipeline, as well as the efficiency of the probe design. Parameters of the various stages in the sequencing process can also be evaluated in order to optimize the experiments. Availability and implementation: CapSim is publicly available under BSD license at https://github.com/Devika1/capsim. Contact: [email protected]. Supplementary information: Supplementary data are available at Bioinformatics online.

BACKGROUND: Tandem repeats comprise significant proportion of the human genome including coding and regulatory regions. They are highly prone to repeat number variation and nucleotide mutation due to their repetitive and unstable nature, making them a major source of genomic variation between individuals. Despite recent advances in high throughput sequencing, analysis of tandem repeats in the context of complex diseases is still hindered by technical limitations. We report a novel targeted sequencing approach, which allows simultaneous analysis of hundreds of repeats. We developed a Bayesian algorithm, namely - GtTR - which combines information from a reference long-read dataset with a short read counting approach to genotype tandem repeats at population scale. PCR sizing analysis was used for validation. RESULTS: We used a PacBio long-read sequenced sample to generate a reference tandem repeat genotype dataset with on average 13% absolute deviation from PCR sizing results. Using this reference dataset GtTR generated estimates of VNTR copy number with accuracy within 95% high posterior density (HPD) intervals of 68 and 83% for capture sequence data and 200X WGS data respectively, improving to 87 and 94% with use of a PCR reference. We show that the genotype resolution increases as a function of depth, such that the median 95% HPD interval lies within 25, 14, 12 and 8% of the its midpoint copy number value for 30X, 200X WGS, 395X and 800X capture sequence data respectively. We validated nine targets by PCR sizing analysis and genotype estimates from sequencing results correlated well with PCR results. CONCLUSIONS: The novel genotyping approach described here presents a new cost-effective method to explore previously unrecognized class of repeat variation in GWAS studies of complex diseases at the population level. Further improvements in accuracy can be obtained by improving accuracy of the reference dataset.

BACKGROUND: Detection of genomic inversions remains challenging. Many existing methods primarily target inzversions with a non repetitive breakpoint, leaving inverted repeat (IR) mediated non-allelic homologous recombination (NAHR) inversions largely unexplored. RESULT: We present npInv, a novel tool specifically for detecting and genotyping NAHR inversion using long read sub-alignment of long read sequencing data. We benchmark npInv with other tools in both simulation and real data. We use npInv to generate a whole-genome inversion map for NA12878 consisting of 30 NAHR inversions (of which 15 are novel), including all previously known NAHR mediated inversions in NA12878 with flanking IR less than 7kb. Our genotyping accuracy on this dataset was 94%. We used PCR to confirm the presence of two of these novel inversions. We show that there is a near linear relationship between the length of flanking IR and the minimum inversion size, without inverted repeats. CONCLUSION: The application of npInv shows high accuracy in both simulation and real data. The results give deeper insight into understanding inversion.

MOTIVATION: The recently released Oxford Nanopore MinION sequencing platform presents many innovative features opening up potential for a range of applications not previously possible. Among these features, the ability to sequence in real-time provides a unique opportunity for many time-critical applications. While many software packages have been developed to analyze its data, there is still a lack of toolkits that support the streaming and real-time analysis of MinION sequencing data. RESULTS: We developed npReader, an open-source software package to facilitate real-time analysis of MinION sequencing data. npReader can simultaneously extract sequence reads and stream them to downstream analysis pipelines while the samples are being sequenced on the MinION device. It provides a command line interface for easy integration into a bioinformatics work flow, as well as a graphical user interface which concurrently displays the statistics of the run. It also provides an application programming interface for development of streaming algorithms in order to fully utilize the extent of nanopore sequencing potential. AVAILABILITY AND IMPLEMENTATION: npReader is written in Java and is freely available at https://github.com/mdcao/npReader CONTACT: [email protected] or [email protected].