The sequences of 150,119 genomes in the UK Biobank.

Detailed knowledge of how diversity in the sequence of the human genome affects phenotypic diversity depends on a comprehensive and reliable characterization of both sequences and phenotypic variation. Over the past decade, insights into this relationship have been obtained from whole-exome sequencing or whole-genome sequencing of large cohorts with rich phenotypic data1,2. Here we describe the analysis of whole-genome sequencing of 150,119 individuals from the UK Biobank3. This constitutes a set of high-quality variants, including 585,040,410 single-nucleotide polymorphisms, representing 7.0% of all possible human single-nucleotide polymorphisms, and 58,707,036 indels. This large set of variants allows us to characterize selection based on sequence variation within a population through a depletion rank score of windows along the genome. Depletion rank analysis shows that coding exons represent a small fraction of regions in the genome subject to strong sequence conservation. We define three cohorts within the UK Biobank: a large British Irish cohort, a smaller African cohort and a South Asian cohort. A haplotype reference panel is provided that allows reliable imputation of most variants carried by three or more sequenced individuals. We identified 895,055 structural variants and 2,536,688 microsatellites, groups of variants typically excluded from large-scale whole-genome sequencing studies. Using this formidable new resource, we provide several examples of trait associations for rare variants with large effects not found previously through studies based on whole-exome sequencing and/or imputation.

Population-scale detection of non-reference sequence variants using colored de Bruijn graphs.

MOTIVATION: With the increasing throughput of sequencing technologies, structural variant (SV) detection has become possible across tens of thousands of genomes. Non-reference sequence (NRS) variants have drawn less attention compared with other types of SVs due to the computational complexity of detecting them. When using short-read data, the detection of NRS variants inevitably involves a de novo assembly which requires high-quality sequence data at high coverage. Previous studies have demonstrated how sequence data of multiple genomes can be combined for the reliable detection of NRS variants. However, the algorithms proposed in these studies have limited scalability to larger sets of genomes. RESULTS: We introduce PopIns2, a tool to discover and characterize NRS variants in many genomes, which scales to considerably larger numbers of genomes than its predecessor PopIns. In this article, we briefly outline the PopIns2 workflow and highlight our novel algorithmic contributions. We developed an entirely new approach for merging contig assemblies of unaligned reads from many genomes into a single set of NRS using a colored de Bruijn graph. Our tests on simulated data indicate that the new merging algorithm ranks among the best approaches in terms of quality and reliability and that PopIns2 shows the best precision for a growing number of genomes processed. Results on the Polaris Diversity Cohort and a set of 1000 Icelandic human genomes demonstrate unmatched scalability for the application on population-scale datasets. AVAILABILITY AND IMPLEMENTATION: The source code of PopIns2 is available from https://github.com/kehrlab/PopIns2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

kallisto, bustools, and kb-python for quantifying bulk, single-cell, and single-nucleus RNA-seq.

The term "RNA-seq" refers to a collection of assays based on sequencing experiments that involve quantifying RNA species from bulk tissue, from single cells, or from single nuclei. The kallisto, bustools, and kb-python programs are free, open-source software tools for performing this analysis that together can produce gene expression quantification from raw sequencing reads. The quantifications can be individualized for multiple cells, multiple samples, or both. Additionally, these tools allow gene expression values to be classified as originating from nascent RNA species or mature RNA species, making this workflow amenable to both cell-based and nucleus-based assays. This protocol describes in detail how to use kallisto and bustools in conjunction with a wrapper, kb-python, to preprocess RNA-seq data.

A comparison of methods for detecting DNA methylation from long-read sequencing of human genomes.

BACKGROUND: Long-read sequencing can enable the detection of base modifications, such as CpG methylation, in single molecules of DNA. The most commonly used methods for long-read sequencing are nanopore developed by Oxford Nanopore Technologies (ONT) and single molecule real-time (SMRT) sequencing developed by Pacific Bioscience (PacBio). In this study, we systematically compare the performance of CpG methylation detection from long-read sequencing. RESULTS: We demonstrate that CpG methylation detection from 7179 nanopore-sequenced DNA samples is highly accurate and consistent with 132 oxidative bisulfite-sequenced (oxBS) samples, isolated from the same blood draws. We introduce quality filters for CpGs that further enhance the accuracy of CpG methylation detection from nanopore-sequenced DNA, while removing at most 30% of CpGs. We evaluate the per-site performance of CpG methylation detection across different genomic features and CpG methylation rates and demonstrate how the latest R10.4 flowcell chemistry and base-calling algorithms improve methylation detection from nanopore sequencing. Additionally, we show how the methylation detection of 50 SMRT-sequenced genomes compares to nanopore sequencing and oxBS. CONCLUSIONS: This study provides the first systematic comparison of CpG methylation detection tools for long-read sequencing methods. We compare two commonly used computational methods for the detection of CpG methylation in a large number of nanopore genomes, including samples sequenced using the latest R10.4 nanopore flowcell chemistry and 50 SMRT sequenced samples. We provide insights into the strengths and limitations of each sequencing method as well as recommendations for standardization and evaluation of tools designed for genome-scale modified base detection using long-read sequencing.

Sequence variants affecting the genome-wide rate of germline microsatellite mutations.

Microsatellites are polymorphic tracts of short tandem repeats with one to six base-pair (bp) motifs and are some of the most polymorphic variants in the genome. Using 6084 Icelandic parent-offspring trios we estimate 63.7 (95% CI: 61.9-65.4) microsatellite de novo mutations (mDNMs) per offspring per generation, excluding one bp repeats motifs (homopolymers) the estimate is 48.2 mDNMs (95% CI: 46.7-49.6). Paternal mDNMs occur at longer repeats than maternal ones, which are in turn larger with a mean size of 3.4 bp vs 3.1 bp for paternal ones. mDNMs increase by 0.97 (95% CI: 0.90-1.04) and 0.31 (95% CI: 0.25-0.37) per year of father's and mother's age at conception, respectively. Here, we find two independent coding variants that associate with the number of mDNMs transmitted to offspring; The minor allele of a missense variant (allele frequency (AF) = 1.9%) in MSH2, a mismatch repair gene, increases transmitted mDNMs from both parents (effect: 13.1 paternal and 7.8 maternal mDNMs). A synonymous variant (AF = 20.3%) in NEIL2, a DNA damage repair gene, increases paternally transmitted mDNMs (effect: 4.4 mDNMs). Thus, the microsatellite mutation rate in humans is in part under genetic control.

BlastFrost: fast querying of 100,000s of bacterial genomes in Bifrost graphs.

BlastFrost is a highly efficient method for querying 100,000s of genome assemblies, building on Bifrost, a dynamic data structure for compacted and colored de Bruijn graphs. BlastFrost queries a Bifrost data structure for sequences of interest and extracts local subgraphs, enabling the identification of the presence or absence of individual genes or single nucleotide sequence variants. We show two examples using Salmonella genomes: finding within minutes the presence of genes in the SPI-2 pathogenicity island in a collection of 926 genomes and identifying single nucleotide polymorphisms associated with fluoroquinolone resistance in three genes among 190,209 genomes. BlastFrost is available at https://github.com/nluhmann/BlastFrost/tree/master/data .

Ratatosk: hybrid error correction of long reads enables accurate variant calling and assembly.

A major challenge to long read sequencing data is their high error rate of up to 15%. We present Ratatosk, a method to correct long reads with short read data. We demonstrate on 5 human genome trios that Ratatosk reduces the error rate of long reads 6-fold on average with a median error rate as low as 0.22 %. SNP calls in Ratatosk corrected reads are nearly 99 % accurate and indel calls accuracy is increased by up to 37 %. An assembly of Ratatosk corrected reads from an Ashkenazi individual yields a contig N50 of 45 Mbp and less misassemblies than a PacBio HiFi reads assembly.

Long-read sequencing of 3,622 Icelanders provides insight into the role of structural variants in human diseases and other traits.

Long-read sequencing (LRS) promises to improve the characterization of structural variants (SVs). We generated LRS data from 3,622 Icelanders and identified a median of 22,636 SVs per individual (a median of 13,353 insertions and 9,474 deletions). We discovered a set of 133,886 reliably genotyped SV alleles and imputed them into 166,281 individuals to explore their effects on diseases and other traits. We discovered an association of a rare deletion in PCSK9 with lower low-density lipoprotein (LDL) cholesterol levels, compared to the population average. We also discovered an association of a multiallelic SV in ACAN with height; we found 11 alleles that differed in the number of a 57-bp-motif repeat and observed a linear relationship between the number of repeats carried and height. These results show that SVs can be accurately characterized at the population scale using LRS data in a genome-wide non-targeted approach and demonstrate how SVs impact phenotypes.

Bifrost: highly parallel construction and indexing of colored and compacted de Bruijn graphs.

Memory consumption of de Bruijn graphs is often prohibitive. Most de Bruijn graph-based assemblers reduce the complexity by compacting paths into single vertices, but this is challenging as it requires the uncompacted de Bruijn graph to be available in memory. We present a parallel and memory-efficient algorithm enabling the direct construction of the compacted de Bruijn graph without producing the intermediate uncompacted graph. Bifrost features a broad range of functions, such as indexing, editing, and querying the graph, and includes a graph coloring method that maps each k-mer of the graph to the genomes it occurs in.Availability https://github.com/pmelsted/bifrost.

Pan-Genome Storage and Analysis Techniques.

Computational pan-genome analysis has emerged from the rapid increase of available genome sequencing data. Starting from a microbial pan-genome, the concept has spread to a variety of species, such as plants or viruses. Characterizing a pan-genome provides insights into intra-species evolution, functions, and diversity. However, researchers face challenges such as processing and maintaining large datasets while providing accurate and efficient analysis approaches. Comparative genomics methods are required for detecting conserved and unique regions between a set of genomes. This chapter gives an overview of tools available for indexing pan-genomes, identifying the sub-regions of a pan-genome and offering a variety of downstream analysis methods. These tools are categorized into two groups, gene-based and sequence-based, according to the pan-genome identification method. We highlight the differences, advantages, and disadvantages between the tools, and provide information about the general workflow, methodology of pan-genome identification, covered functionalities, usability and availability of the tools.

Dynamic Alignment-Free and Reference-Free Read Compression.

The advent of high throughput sequencing (HTS) technologies raises a major concern about storage and transmission of data produced by these technologies. In particular, large-scale sequencing projects generate an unprecedented volume of genomic sequences ranging from tens to several thousands of genomes per species. These collections contain highly similar and redundant sequences, also known as pangenomes. The ideal way to represent and transfer pangenomes is through compression. A number of HTS-specific compression tools have been developed to reduce the storage and communication costs of HTS data, yet none of them is designed to process a pangenome. In this article, we present dynamic alignment-free and reference-free read compression (DARRC), a new alignment-free and reference-free compression method. It addresses the problem of pangenome compression by encoding the sequences of a pangenome as a guided de Bruijn graph. The novelty of this method is its ability to incrementally update DARRC archives with new genome sequences without full decompression of the archive. DARRC can compress both single-end and paired-end read sequences of any length using all symbols of the IUPAC nucleotide code. On a large Pseudomonas aeruginosa data set, our method outperforms all other tested tools. It provides a 30% compression ratio improvement in single-end mode compared with the best performing state-of-the-art HTS-specific compression method in our experiments.

Bloom Filter Trie: an alignment-free and reference-free data structure for pan-genome storage.

BACKGROUND: High throughput sequencing technologies have become fast and cheap in the past years. As a result, large-scale projects started to sequence tens to several thousands of genomes per species, producing a high number of sequences sampled from each genome. Such a highly redundant collection of very similar sequences is called a pan-genome. It can be transformed into a set of sequences "colored" by the genomes to which they belong. A colored de Bruijn graph (C-DBG) extracts from the sequences all colored k-mers, strings of length k, and stores them in vertices. RESULTS: In this paper, we present an alignment-free, reference-free and incremental data structure for storing a pan-genome as a C-DBG: the bloom filter trie (BFT). The data structure allows to store and compress a set of colored k-mers, and also to efficiently traverse the graph. Bloom filter trie was used to index and query different pangenome datasets. Compared to another state-of-the-art data structure, BFT was up to two times faster to build while using about the same amount of main memory. For querying k-mers, BFT was about 52-66 times faster while using about 5.5-14.3 times less memory. CONCLUSION: We present a novel succinct data structure called the Bloom Filter Trie for indexing a pan-genome as a colored de Bruijn graph. The trie stores k-mers and their colors based on a new representation of vertices that compress and index shared substrings. Vertices use basic data structures for lightweight substrings storage as well as Bloom filters for efficient trie and graph traversals. Experimental results prove better performance compared to another state-of-the-art data structure. AVAILABILITY: https://www.github.com/GuillaumeHolley/BloomFilterTrie.