High-throughput sequencing technologies promise to transform the fields of genetics and comparative biology by delivering tens of thousands of genomes in the near future. Although it is feasible to ...construct de novo genome assemblies in a few months, there has been relatively little attention to what is lost by sole application of short sequence reads. We compared the recent de novo assemblies using the short oligonucleotide analysis package (SOAP), generated from the genomes of a Han Chinese individual and a Yoruban individual, to experimentally validated genomic features. We found that de novo assemblies were 16.2% shorter than the reference genome and that 420.2 megabase pairs of common repeats and 99.1% of validated duplicated sequences were missing from the genome. Consequently, over 2,377 coding exons were completely missing. We conclude that high-quality sequencing approaches must be considered in conjunction with high-throughput sequencing for comparative genomics analyses and studies of genome evolution.
Here, we present a comprehensive analysis on the reproducibility of computational characterization of genomic variants using high throughput sequencing data. We reanalyzed the same datasets twice, ...using the same tools with the same parameters, where we only altered the order of reads in the input (i.e. FASTQ file). Reshuffling caused the reads from repetitive regions being mapped to different locations in the second alignment, and we observed similar results when we only applied a scatter/gather approach for read mapping-without prior shuffling. Our results show that, some of the most common variation discovery algorithms do not handle the ambiguous read mappings accurately when random locations are selected. In addition, we also observed that even when the exact same alignment is used, the GATK HaplotypeCaller generates slightly different call sets, which we pinpoint to the variant filtration step. We conclude that, algorithms at each step of genomic variation discovery and characterization need to treat ambiguous mappings in a deterministic fashion to ensure full replication of results.
Code, scripts and the generated VCF files are available at DOI:10.5281/zenodo.32611.
calkan@cs.bilkent.edu.tr
Supplementary data are available at Bioinformatics online.
Seed location filtering is critical in DNA read mapping, a process where billions of DNA fragments (reads) sampled from a donor are mapped onto a reference genome to identify genomic variants of the ...donor. State-of-the-art read mappers 1) quickly generate possible mapping locations for seeds (i.e., smaller segments) within each read, 2) extract reference sequences at each of the mapping locations, and 3) check similarity between each read and its associated reference sequences with a computationally-expensive algorithm (i.e., sequence alignment) to determine the origin of the read. A seed location filter comes into play before alignment, discarding seed locations that alignment would deem a poor match. The ideal seed location filter would discard all poor match locations prior to alignment such that there is no wasted computation on unnecessary alignments.
We propose a novel seed location filtering algorithm, GRIM-Filter, optimized to exploit 3D-stacked memory systems that integrate computation within a logic layer stacked under memory layers, to perform processing-in-memory (PIM). GRIM-Filter quickly filters seed locations by 1) introducing a new representation of coarse-grained segments of the reference genome, and 2) using massively-parallel in-memory operations to identify read presence within each coarse-grained segment. Our evaluations show that for a sequence alignment error tolerance of 0.05, GRIM-Filter 1) reduces the false negative rate of filtering by 5.59x-6.41x, and 2) provides an end-to-end read mapper speedup of 1.81x-3.65x, compared to a state-of-the-art read mapper employing the best previous seed location filtering algorithm.
GRIM-Filter exploits 3D-stacked memory, which enables the efficient use of processing-in-memory, to overcome the memory bandwidth bottleneck in seed location filtering. We show that GRIM-Filter significantly improves the performance of a state-of-the-art read mapper. GRIM-Filter is a universal seed location filter that can be applied to any read mapper. We hope that our results provide inspiration for new works to design other bioinformatics algorithms that take advantage of emerging technologies and new processing paradigms, such as processing-in-memory using 3D-stacked memory devices.
The 1000 Genomes Project (1kGP) is the largest fully open resource of whole-genome sequencing (WGS) data consented for public distribution without access or use restrictions. The final, phase 3 ...release of the 1kGP included 2,504 unrelated samples from 26 populations and was based primarily on low-coverage WGS. Here, we present a high-coverage 3,202-sample WGS 1kGP resource, which now includes 602 complete trios, sequenced to a depth of 30X using Illumina. We performed single-nucleotide variant (SNV) and short insertion and deletion (INDEL) discovery and generated a comprehensive set of structural variants (SVs) by integrating multiple analytic methods through a machine learning model. We show gains in sensitivity and precision of variant calls compared to phase 3, especially among rare SNVs as well as INDELs and SVs spanning frequency spectrum. We also generated an improved reference imputation panel, making variants discovered here accessible for association studies.
Structural variants are implicated in numerous diseases and make up the majority of varying nucleotides among human genomes. Here we describe an integrated set of eight structural variant classes ...comprising both balanced and unbalanced variants, which we constructed using short-read DNA sequencing data and statistically phased onto haplotype blocks in 26 human populations. Analysing this set, we identify numerous gene-intersecting structural variants exhibiting population stratification and describe naturally occurring homozygous gene knockouts that suggest the dispensability of a variety of human genes. We demonstrate that structural variants are enriched on haplotypes identified by genome-wide association studies and exhibit enrichment for expression quantitative trait loci. Additionally, we uncover appreciable levels of structural variant complexity at different scales, including genic loci subject to clusters of repeated rearrangement and complex structural variants with multiple breakpoints likely to have formed through individual mutational events. Our catalogue will enhance future studies into structural variant demography, functional impact and disease association.
Aligning sequencing reads onto a reference is an essential step of the majority of genomic analysis pipelines. Computational algorithms for read alignment have evolved in accordance with ...technological advances, leading to today's diverse array of alignment methods. We provide a systematic survey of algorithmic foundations and methodologies across 107 alignment methods, for both short and long reads. We provide a rigorous experimental evaluation of 11 read aligners to demonstrate the effect of these underlying algorithms on speed and efficiency of read alignment. We discuss how general alignment algorithms have been tailored to the specific needs of various domains in biology.
New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution and comprehensiveness. To help translate these methods to ...routine research and clinical practice, we developed a sequence-resolved benchmark set for identification of both false-negative and false-positive germline large insertions and deletions. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle Consortium integrated 19 sequence-resolved variant calling methods from diverse technologies. The final benchmark set contains 12,745 isolated, sequence-resolved insertion (7,281) and deletion (5,464) calls ≥50 base pairs (bp). The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.51 Gbp and 5,262 insertions and 4,095 deletions supported by ≥1 diploid assembly. We demonstrate that the benchmark set reliably identifies false negatives and false positives in high-quality SV callsets from short-, linked- and long-read sequencing and optical mapping.
We present a DNA library preparation method that has allowed us to reconstruct a high-coverage (30×) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome ...allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of "missing evolution" in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans.
Recent studies show that along with single nucleotide polymorphisms and small indels, larger structural variants among human individuals are common. The Human Genome Structural Variation Project aims ...to identify and classify deletions, insertions, and inversions (>5 Kbp) in a small number of normal individuals with a fosmid-based paired-end sequencing approach using traditional sequencing technologies. The realization of new ultra-high-throughput sequencing platforms now makes it feasible to detect the full spectrum of genomic variation among many individual genomes, including cancer patients and others suffering from diseases of genomic origin. Unfortunately, existing algorithms for identifying structural variation (SV) among individuals have not been designed to handle the short read lengths and the errors implied by the "next-gen" sequencing (NGS) technologies. In this paper, we give combinatorial formulations for the SV detection between a reference genome sequence and a next-gen-based, paired-end, whole genome shotgun-sequenced individual. We describe efficient algorithms for each of the formulations we give, which all turn out to be fast and quite reliable; they are also applicable to all next-gen sequencing methods (Illumina, 454 Life Sciences Roche, ABI SOLiD, etc.) and traditional capillary sequencing technology. We apply our algorithms to identify SV among individual genomes very recently sequenced by Illumina technology.
With the introduction of next-generation sequencing (NGS) technologies, we are facing an exponential increase in the amount of genomic sequence data. The success of all medical and genetic ...applications of next-generation sequencing critically depends on the existence of computational techniques that can process and analyze the enormous amount of sequence data quickly and accurately. Unfortunately, the current read mapping algorithms have difficulties in coping with the massive amounts of data generated by NGS.We propose a new algorithm, FastHASH, which drastically improves the performance of the seed-and-extend type hash table based read mapping algorithms, while maintaining the high sensitivity and comprehensiveness of such methods. FastHASH is a generic algorithm compatible with all seed-and-extend class read mapping algorithms. It introduces two main techniques, namely Adjacency Filtering, and Cheap K-mer Selection.We implemented FastHASH and merged it into the codebase of the popular read mapping program, mrFAST. Depending on the edit distance cutoffs, we observed up to 19-fold speedup while still maintaining 100% sensitivity and high comprehensiveness.
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