The Sequence Alignment/Map format and SAMtools

Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments

The variant call format and VCFtools

Summary: The variant call format (VCF) is a generic format for storing DNA polymorphism data such as SNPs, insertions, deletions and structural variants, together with rich annotations. VCF is usually stored in a compressed manner and can be indexed for fast data retrieval of variants from a range of positions on the reference genome. The format was developed for the 1000 Genomes Project, and has also been adopted by other projects such as UK10K, dbSNP and the NHLBI Exome Project. VCFtools is a software suite that implements various utilities for processing VCF files, including validation, merging, comparing and also provides a general Perl API

Whole genome resequencing of a laboratory-adapted Drosophila melanogaster population sample

As part of a study into the molecular genetics of sexually dimorphic complex traits, we used high-throughput sequencing to obtain data on genomic variation in an outbred laboratory-adapted fruit fly (Drosophila melanogaster) population. We successfully resequenced the whole genome of 220 hemiclonal females that were heterozygous for the same Berkeley reference line genome (BDGP6/dm6), and a unique haplotype from the outbred base population (LHM). The use of a static and known genetic background enabled us to obtain sequences from whole-genome phased haplotypes. We used a BWA-Picard-GATK pipeline for mapping sequence reads to the dm6 reference genome assembly, at a median depth-of coverage of 31X, and have made the resulting data publicly-available in the NCBI Short Read Archive (Accession number SRP058502). We used Haplotype Caller to discover and genotype 1,726,931 small genomic variants (SNPs and indels, <200bp). Additionally we detected and genotyped 167 large structural variants (1-100Kb in size) using GenomeStrip/2.0. Sequence and genotype data are publicly-available at the corresponding NCBI databases: Short Read Archive, dbSNP and dbVar (BioProject PRJNA282591). We have also released the unfiltered genotype data, and the code and logs for data processing and summary statistics

Contribution of retrotransposition to developmental disorders.

Mobile genetic Elements (MEs) are segments of DNA which can copy themselves and other transcribed sequences through the process of retrotransposition (RT). In humans several disorders have been attributed to RT, but the role of RT in severe developmental disorders (DD) has not yet been explored. Here we identify RT-derived events in 9738 exome sequenced trios with DD-affected probands. We ascertain 9 de novo MEs, 4 of which are likely causative of the patient's symptoms (0.04%), as well as 2 de novo gene retroduplications. Beyond identifying likely diagnostic RT events, we estimate genome-wide germline ME mutation rate and selective constraint and demonstrate that coding RT events have signatures of purifying selection equivalent to those of truncating mutations. Overall, our analysis represents a comprehensive interrogation of the impact of retrotransposition on protein coding genes and a framework for future evolutionary and disease studies

Mapping Copy Number Variation by Population Scale Genome Sequencing

Genomic structural variants (SVs) are abundant in humans, differing from other forms of variation in extent, origin and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (that is, copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analysing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.Organismic and Evolutionary Biolog

Using population admixture to help complete maps of the human genome

Tens of millions of base pairs of euchromatic human genome sequence, including many protein-coding genes, have no known location in the human genome. We describe an approach for localizing the human genome's missing pieces by utilizing the patterns of genome sequence variation created by population admixture. We mapped the locations of 70 scaffolds spanning four million base pairs of the human genome's unplaced euchromatic sequence, including more than a dozen protein-coding genes, and identified eight large novel inter-chromosomal segmental duplications. We find that most of these sequences are hidden in the genome's heterochromatin, particularly its pericentromeric regions. Many cryptic, pericentromeric genes are expressed in RNA and have been maintained intact for millions of years while their expression patterns diverged from those of paralogous genes elsewhere in the genome. We describe how knowledge of the locations of these sequences can inform disease association and genome biology studies