Long-read human genome sequencing and its applications

Logsdon, Glennis A.; Vollger, Mitchell R.; Eichler, Evan E.

doi:10.1038/s41576-020-0236-x

Cited by 680 publications

(635 citation statements)

References 145 publications

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“…Most of the SVs reported in this study are deletions that are unlikely to be of evolutionary benefit. However, this limitation probably reflects the use of short read sequences that have been reported to miss most of the SVs larger than 2kb including duplications and inversions (40). In fact, a prior study that compared the assembled genomes of two virtually identical rice accessions undergoing independent mPing bursts: HEG4 and EG4, detected an inversion of ~120kb with mPings at the breakpoints (9).…”

Section: Discussionmentioning

confidence: 99%

Genomic diversity generated by a transposable element burst in a rice recombinant inbred population

Chen

Robb

et al. 2020

Preprint

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Genomes of all characterized higher eukaryotes harbor examples of transposable element (TE) bursts - the rapid amplification of TE copies throughout a genome. Despite their prevalence, understanding how bursts diversify genomes requires the characterization of actively transposing TEs before insertion sites and structural rearrangements have been obscured by selection acting over evolutionary time. In this study rice recombinant inbred lines (RILs), generated by crossing a bursting accession and the reference Nipponbare accession were exploited to characterize the spread of the very active Ping/mPing family through a small population and the resulting impact on genome diversity. Comparative sequence analysis of 272 individuals led to the identification of over 14,000 new insertions of the mPing miniature inverted-repeat transposable element (MITE) with no evidence for silencing of the transposase-encoding Ping element. In addition to new insertions, Ping-encoded transposase was found to preferentially catalyze the excision of mPing loci tightly linked to a second mPing insertion. Similarly, structural variations, including deletion of rice exons or regulatory regions, were enriched for those with breakpoints at one or both ends of linked mPing elements. Taken together, these results indicate that structural variations are generated during a TE burst as transposase catalyzes both the high copy numbers needed to distribute linked elements throughout the genome and the DNA cuts at the TE ends known to dramatically increase the frequency of recombination.

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Section: Discussionmentioning

confidence: 99%

Genomic diversity generated by a transposable element burst in a rice recombinant inbred population

Chen

Robb

et al. 2020

Preprint

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“…CHM13 PacBio HiFi and ONT data were aligned to the entire chromosome 8 assembly via pbmm2 (v1.1.0) (for PacBio data; https://github.com/PacificBiosciences/pbmm2) or Winnowmap 55 (v1.0) (for ONT data) to identify large collapses or misassemblies. Although the ONT-based scaffolds are structurally accurate, they are only 87-98% accurate at the base level due to base-calling errors in the raw ONT reads 13 . Therefore, we sought to improve the base accuracy of the sequence scaffolds by replacing the ONT sequences with PacBio HiFi contigs assembled from the CHM13 genome 11 , which have a consensus accuracy greater than 99.99% 11 .…”

Section: Targeted Sequence Assemblymentioning

confidence: 99%

“…The advent of long-read sequencing technologies and associated algorithms have now made it possible to systematically assemble these regions from native DNA for the first time [11][12][13] . In addition, the use of DNA from complete hydatidiform moles (CHMs) to serve as reference genomes has greatly simplified sequence resolution of these complex regions.…”

Section: Introductionmentioning

confidence: 99%

The structure, function, and evolution of a complete human chromosome 8

Logsdon

Hsieh

Mao

et al. 2020

Preprint

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The complete assembly of each human chromosome is essential for understanding human biology and evolution. Using complementary long-read sequencing technologies, we complete the first linear assembly of a human autosome, chromosome 8. Our assembly resolves the sequence of five previously long-standing gaps, including a 2.08 Mbp centromeric α-satellite array, a 644 kbp defensin copy number polymorphism important for disease risk, and an 863 kbp variable number tandem repeat at chromosome 8q21.2 that can function as a neocentromere. We show that the centromeric α-satellite array is generally methylated except for a 73 kbp hypomethylated region of diverse higher-order α-satellite enriched with CENP-A nucleosomes, consistent with the location of the kinetochore. Using a dual long-read sequencing approach, we complete the assembly of the orthologous chromosome 8 centromeric regions in chimpanzee, orangutan, and macaque for the first time to reconstruct its evolutionary history. Comparative and phylogenetic analyses show that the higher-order α-satellite structure evolved specifically in the great ape ancestor, and the centromeric region evolved with a layered symmetry, with more ancient higher-order repeats located at the periphery adjacent to monomeric α-satellites. We estimate that the mutation rate of centromeric satellite DNA is accelerated at least 2.2-fold, and this acceleration extends beyond the higher-order α-satellite into the flanking sequence.

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“…Oxford Nanopore Technologies (ONT) and Pacific Bioscience (PacBio) are LRS platforms [Logsdon et al, 2020] that produce long sequence reads ranging from 10 3 to 10 6 bases with an error rate up to 15% [Rang et al, 2018]. The high error rate of LRS reads is in part compensated by their lengths which increase their mapping accuracy, making LRS suitable for numerous applications in all fields of genomics.…”

Section: Introductionmentioning

confidence: 99%

Ratatosk – Hybrid error correction of long reads enables accurate variant calling and assembly

Holley

Beyter

Ingimundardóttir

et al. 2020

Preprint

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Motivation: Long Read Sequencing (LRS) technologies are becoming essential to complement Short Read Sequencing (SRS) technologies for routine whole genome sequencing. LRS platforms produce DNA fragment reads, thousands to millions bases long, allowing the resolution of numerous uncertainties left by SRS reads for genome reconstruction and analysis. In particular, LRS characterizes long and complex structural variants undetected by SRS due to short read length. Furthermore, assemblies produced with LRS reads are considerably more contiguous than with SRS while spanning previously inaccessible telomeric and centromeric regions. However, a major challenge to LRS reads adoption is their much higher error rate than SRS of up to 15%, introducing obstacles in downstream analysis pipelines. Results: We present Ratatosk, a new error correction method for erroneous long reads based on a compacted and colored de Bruijn graph built from accurate short reads. Short and long reads color paths in the graph while vertices are annotated with candidate Single Nucleotide Polymorphisms. Long reads are subsequently anchored to the graph using exact and inexact k-mer matches to find paths corresponding to corrected sequences. We demonstrate that Ratatosk can reduce the raw error rate of Oxford Nanopore reads 6-fold on average with a median error rate as low as 0.28%. Ratatosk corrected data maintain nearly 99% accurate SNP calls and increase indel call accuracy by up to about 40% compared to the raw data. An assembly of the Ashkenazi individual HG002 created from Ratatosk corrected Oxford Nanopore reads yields a contig N50 of 43.22 Mbp and less misassemblies than an assembly created from PacBio HiFi reads. Availability: https://github.com/DecodeGenetics/Ratatosk Contact: guillaume{dot}holley{at}decode{dot}is

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Long-read human genome sequencing and its applications

Cited by 680 publications

References 145 publications

Genomic diversity generated by a transposable element burst in a rice recombinant inbred population

Genomic diversity generated by a transposable element burst in a rice recombinant inbred population

The structure, function, and evolution of a complete human chromosome 8

Ratatosk – Hybrid error correction of long reads enables accurate variant calling and assembly

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