Recent long-read assemblies often exceed the quality and completeness of available reference genomes, making validation challenging. Here we present Merqury, a novel tool for reference-free assembly ...evaluation based on efficient k-mer set operations. By comparing k-mers in a de novo assembly to those found in unassembled high-accuracy reads, Merqury estimates base-level accuracy and completeness. For trios, Merqury can also evaluate haplotype-specific accuracy, completeness, phase block continuity, and switch errors. Multiple visualizations, such as k-mer spectrum plots, can be generated for evaluation. We demonstrate on both human and plant genomes that Merqury is a fast and robust method for assembly validation.
Long-read sequencing and novel long-range assays have revolutionized de novo genome assembly by automating the reconstruction of reference-quality genomes. In particular, Hi-C sequencing is becoming ...an economical method for generating chromosome-scale scaffolds. Despite its increasing popularity, there are limited open-source tools available. Errors, particularly inversions and fusions across chromosomes, remain higher than alternate scaffolding technologies. We present a novel open-source Hi-C scaffolder that does not require an a priori estimate of chromosome number and minimizes errors by scaffolding with the assistance of an assembly graph. We demonstrate higher accuracy than the state-of-the-art methods across a variety of Hi-C library preparations and input assembly sizes. The Python and C++ code for our method is openly available at https://github.com/machinegun/SALSA.
Long-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. However, given the relatively high error rates ...of such technologies, efficient and accurate assembly of large repeats and closely related haplotypes remains challenging. We address these issues with Canu, a successor of Celera Assembler that is specifically designed for noisy single-molecule sequences. Canu introduces support for nanopore sequencing, halves depth-of-coverage requirements, and improves assembly continuity while simultaneously reducing runtime by an order of magnitude on large genomes versus Celera Assembler 8.2. These advances result from new overlapping and assembly algorithms, including an adaptive overlapping strategy based on
weighted MinHash and a sparse assembly graph construction that avoids collapsing diverged repeats and haplotypes. We demonstrate that Canu can reliably assemble complete microbial genomes and near-complete eukaryotic chromosomes using either Pacific Biosciences (PacBio) or Oxford Nanopore technologies and achieves a contig NG50 of >21 Mbp on both human and
PacBio data sets. For assembly structures that cannot be linearly represented, Canu provides graph-based assembly outputs in graphical fragment assembly (GFA) format for analysis or integration with complementary phasing and scaffolding techniques. The combination of such highly resolved assembly graphs with long-range scaffolding information promises the complete and automated assembly of complex genomes.
Complete and accurate genome assemblies form the basis of most downstream genomic analyses and are of critical importance. Recent genome assembly projects have relied on a combination of noisy ...long-read sequencing and accurate short-read sequencing, with the former offering greater assembly continuity and the latter providing higher consensus accuracy. The recently introduced Pacific Biosciences (PacBio) HiFi sequencing technology bridges this divide by delivering long reads (>10 kbp) with high per-base accuracy (>99.9%). Here we present HiCanu, a modification of the Canu assembler designed to leverage the full potential of HiFi reads via homopolymer compression, overlap-based error correction, and aggressive false overlap filtering. We benchmark HiCanu with a focus on the recovery of haplotype diversity, major histocompatibility complex (MHC) variants, satellite DNAs, and segmental duplications. For diploid human genomes sequenced to 30× HiFi coverage, HiCanu achieved superior accuracy and allele recovery compared to the current state of the art. On the effectively haploid CHM13 human cell line, HiCanu achieved an NG50 contig size of 77 Mbp with a per-base consensus accuracy of 99.999% (QV50), surpassing recent assemblies of high-coverage, ultralong Oxford Nanopore Technologies (ONT) reads in terms of both accuracy and continuity. This HiCanu assembly correctly resolves 337 out of 341 validation BACs sampled from known segmental duplications and provides the first preliminary assemblies of nine complete human centromeric regions. Although gaps and errors still remain within the most challenging regions of the genome, these results represent a significant advance toward the complete assembly of human genomes.
Single-molecule sequencing instruments can generate multikilobase sequences with the potential to greatly improve genome and transcriptome assembly. However, the error rates of single-molecule reads ...are high, which has limited their use thus far to resequencing bacteria. To address this limitation, we introduce a correction algorithm and assembly strategy that uses short, high-fidelity sequences to correct the error in single-molecule sequences. We demonstrate the utility of this approach on reads generated by a PacBio RS instrument from phage, prokaryotic and eukaryotic whole genomes, including the previously unsequenced genome of the parrot Melopsittacus undulatus, as well as for RNA-Seq reads of the corn (Zea mays) transcriptome. Our long-read correction achieves >99.9% base-call accuracy, leading to substantially better assemblies than current sequencing strategies: in the best example, the median contig size was quintupled relative to high-coverage, second-generation assemblies. Greater gains are predicted if read lengths continue to increase, including the prospect of single-contig bacterial chromosome assembly.
Abstract
Motivation
In this era of exponential data growth, minimizer sampling has become a standard algorithmic technique for rapid genome sequence comparison. This technique yields a sub-linear ...representation of sequences, enabling their comparison in reduced space and time. A key property of the minimizer technique is that if two sequences share a substring of a specified length, then they can be guaranteed to have a matching minimizer. However, because the k-mer distribution in eukaryotic genomes is highly uneven, minimizer-based tools (e.g. Minimap2, Mashmap) opt to discard the most frequently occurring minimizers from the genome to avoid excessive false positives. By doing so, the underlying guarantee is lost and accuracy is reduced in repetitive genomic regions.
Results
We introduce a novel weighted-minimizer sampling algorithm. A unique feature of the proposed algorithm is that it performs minimizer sampling while considering a weight for each k-mer; i.e. the higher the weight of a k-mer, the more likely it is to be selected. By down-weighting frequently occurring k-mers, we are able to meet both objectives: (i) avoid excessive false-positive matches and (ii) maintain the minimizer match guarantee. We tested our algorithm, Winnowmap, using both simulated and real long-read data and compared it to a state-of-the-art long read mapper, Minimap2. Our results demonstrate a reduction in the mapping error-rate from 0.14% to 0.06% in the recently finished human X chromosome (154.3 Mbp), and from 3.6% to 0% within the highly repetitive X centromere (3.1 Mbp). Winnowmap improves mapping accuracy within repeats and achieves these results with sparser sampling, leading to better index compression and competitive runtimes.
Availability and implementation
Winnowmap is built on top of the Minimap2 codebase and is available at https://github.com/marbl/winnowmap.
Complex allelic variation hampers the assembly of haplotype-resolved sequences from diploid genomes. We developed trio binning, an approach that simplifies haplotype assembly by resolving allelic ...variation before assembly. In contrast with prior approaches, the effectiveness of our method improved with increasing heterozygosity. Trio binning uses short reads from two parental genomes to first partition long reads from an offspring into haplotype-specific sets. Each haplotype is then assembled independently, resulting in a complete diploid reconstruction. We used trio binning to recover both haplotypes of a diploid human genome and identified complex structural variants missed by alternative approaches. We sequenced an F1 cross between the cattle subspecies Bos taurus taurus and Bos taurus indicus and completely assembled both parental haplotypes with NG50 haplotig sizes of >20 Mb and 99.998% accuracy, surpassing the quality of current cattle reference genomes. We suggest that trio binning improves diploid genome assembly and will facilitate new studies of haplotype variation and inheritance.
There is much interest in characterizing the variation in a human individual, because this may elucidate what contributes significantly to a person's phenotype, thereby enabling personalized ...genomics. We focus here on the variants in a person's 'exome,' which is the set of exons in a genome, because the exome is believed to harbor much of the functional variation. We provide an analysis of the approximately 12,500 variants that affect the protein coding portion of an individual's genome. We identified approximately 10,400 nonsynonymous single nucleotide polymorphisms (nsSNPs) in this individual, of which approximately 15-20% are rare in the human population. We predict approximately 1,500 nsSNPs affect protein function and these tend be heterozygous, rare, or novel. Of the approximately 700 coding indels, approximately half tend to have lengths that are a multiple of three, which causes insertions/deletions of amino acids in the corresponding protein, rather than introducing frameshifts. Coding indels also occur frequently at the termini of genes, so even if an indel causes a frameshift, an alternative start or stop site in the gene can still be used to make a functional protein. In summary, we reduced the set of approximately 12,500 nonsilent coding variants by approximately 8-fold to a set of variants that are most likely to have major effects on their proteins' functions. This is our first glimpse of an individual's exome and a snapshot of the current state of personalized genomics. The majority of coding variants in this individual are common and appear to be functionally neutral. Our results also indicate that some variants can be used to improve the current NCBI human reference genome. As more genomes are sequenced, many rare variants and non-SNP variants will be discovered. We present an approach to analyze the coding variation in humans by proposing multiple bioinformatic methods to hone in on possible functional variation.
Presented here is a genome sequence of an individual human. It was produced from approximately 32 million random DNA fragments, sequenced by Sanger dideoxy technology and assembled into 4,528 ...scaffolds, comprising 2,810 million bases (Mb) of contiguous sequence with approximately 7.5-fold coverage for any given region. We developed a modified version of the Celera assembler to facilitate the identification and comparison of alternate alleles within this individual diploid genome. Comparison of this genome and the National Center for Biotechnology Information human reference assembly revealed more than 4.1 million DNA variants, encompassing 12.3 Mb. These variants (of which 1,288,319 were novel) included 3,213,401 single nucleotide polymorphisms (SNPs), 53,823 block substitutions (2-206 bp), 292,102 heterozygous insertion/deletion events (indels)(1-571 bp), 559,473 homozygous indels (1-82,711 bp), 90 inversions, as well as numerous segmental duplications and copy number variation regions. Non-SNP DNA variation accounts for 22% of all events identified in the donor, however they involve 74% of all variant bases. This suggests an important role for non-SNP genetic alterations in defining the diploid genome structure. Moreover, 44% of genes were heterozygous for one or more variants. Using a novel haplotype assembly strategy, we were able to span 1.5 Gb of genome sequence in segments >200 kb, providing further precision to the diploid nature of the genome. These data depict a definitive molecular portrait of a diploid human genome that provides a starting point for future genome comparisons and enables an era of individualized genomic information.
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