<i>De novo</i>assembly of two Swedish genomes reveals missing segments from the human GRCh38 reference and improves variant calling of population-scale sequencing data

Ameur, Adam; Che, Huiwen; Martin, Marcel; Bunikis, Ignas; Dahlberg, Johan; Höijer, Ida; Häggqvist, Susana; Vezzi, Francesco; Nordlund, Jessica; Olason, Pall I.; Feuk, Lars; Gyllensten, Ulf

doi:10.1101/267062

Cited by 14 publications

(11 citation statements)

References 39 publications

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“…Human long-read assemblies have been shown to contain several megabases (Mbs) of novel sequences or alternative haplotypes with high diversity from the GRCh38 reference 35,36 . We were interested to determine whether any additional off-targets could be detected in such "dark matter" regions of the HEK293 genome.…”

Section: Searching For Grna Binding In "Dark Matter" Regions Of the Hmentioning

confidence: 99%

Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

Höijer

Johansson

Gudmundsson

et al. 2020

Preprint

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A much-debated concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target sites and activity is challenging. Here we present SMRT-OTS and Nano-OTS, two amplification-free long-read sequencing protocols for detection of gRNA driven digestion of genomic DNA by Cas9. The methods were assessed using the human cell line HEK293, which was first re-sequenced at 18x coverage using highly accurate (HiFi) SMRT reads to get a detailed view of all on-and off-target binding regions. We then applied SMRT-OTS and Nano-OTS to investigate the specificity of three different gRNAs, resulting in a set of 55 highconfidence gRNA binding sites identified by both methods. Twenty-five (45%) of these sites were not reported by off-target prediction software, either because they contained four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. We further discovered that a heterozygous SNP can cause allele-specific gRNA binding. Finally, by performing a de novo genome assembly of the HiFi reads, we were able to re-discover 98.7% of the gRNA binding sites without any prior information about the human reference genome. This suggests that CRISPR-Cas9 off-target sites can be efficiently mapped also in organisms where the genome sequence is unknown. In conclusion, the amplificationfree sequencing protocols revealed many gRNA binding sites in vitro that would be difficult to predict based on gRNA sequence alignment to a reference. Nevertheless, it is still unknown whether in vivo off-target editing would occur at these sites.

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Section: Searching For Grna Binding In "Dark Matter" Regions Of the Hmentioning

confidence: 99%

Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

Höijer

Johansson

Gudmundsson

et al. 2020

Preprint

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“…With an average of 70.2 Gb per PromethION flow cell, we obtained substantially higher yields than the most recently published long-read human genomes. Generation of a 91.2 Gb genome on MinION (ONT) required 39 flow cells (2.3 Gb per flow cell on average) 42 , and 225 SMRTcells were required to produce a 236 Gb genome on a PacBio RSII instrument (1.0 Gb per SMRTcell) 43 . While we show that a single sequencing run is sufficient for long-read human genome sequencing, this study highlights several technical aspects that influence final yield.…”

Section: Promethion Sequencingmentioning

confidence: 99%

Accurate characterization of expanded tandem repeat length and sequence through whole genome long-read sequencing on PromethION

Roeck

Coster

Bossaerts

et al. 2018

Preprint

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De Roeck et al., PromethION sequencing and tandem repeat characterization 2 Abstract Tandem repeats (TRs) can cause disease through their length, sequence motif interruptions, and nucleotide modifications. For many TRs, however, these features are very difficult -if not impossible -to assess, requiring low-throughput and labor-intensive assays. One example is a VNTR in ABCA7 for which we recently discovered that expanded alleles strongly increase risk of Alzheimer's disease. Here, we investigated the potential of long-read whole genome sequencing to surmount these challenges, using the high-throughput PromethION platform from Oxford Nanopore Technologies. To overcome the limitations of conventional base calling and alignment, we developed an algorithm to study the TR size and sequence directly on raw PromethION current data.We report the long-read sequencing of multiple human genomes (n = 11) using only a single sequencing run and flow cell per individual. With the use of fresh DNA extractions, DNA shearing to approximately 20kb and size selection, we obtained an average output of 70 gigabases (Gb) per flow cell, corresponding to a 21x genome coverage, and a maximum yield of 98 Gb (30x genome coverage). All ABCA7 VNTR alleles, including expansions up to 10,000 bases, were spanned by long sequencing reads, validated by Southern blotting. Classical approaches of TR length estimation suffered from low accuracy, low precision, DNA strand effects and/or inability to call pathogenic repeat expansions. In contrast, our novel NanoSatellite algorithm, which circumvents base calling by using dynamic time warping on raw PromethION current data, achieved more than 90% accuracy and high precision (5.6% relative standard deviation) of TR length estimation, and detected all clinically relevant repeat expansions. In addition, we identified alternative TR sequence motifs with high consistency, allowing determination of TR sequence and distinction of VNTR alleles with homozygous length.In conclusion, we validated the robustness of single-experiment whole genome long-read sequencing on PromethION, a prerequisite for application of long-read sequencing in the clinic. In addition, we outperformed Southern blotting, enabling improved characterization of the role of expanded ABCA7 VNTR alleles in Alzheimer's disease, and opening new opportunities for TR research.

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“…sequencers from Oxford Nanopore Technology (ONT) and Pacific Biosciences (PacBio), can read DNA sequences that are orders of magnitude longer than those of second generation sequencing. With longer read lengths, it makes de novo assembly relatively easier and we can generate more contiguous assemblies [6][7][8][9][10][11][12][13] . The de novo reconstruction of a genome reduces the dependence on using a reference as prior information.…”

Section: Introductionmentioning

confidence: 99%

“…A re-sequencing approach depending on a reference may not be effective to explore those genomic structures that are deviated from the references significantly. Recent studies with directly human genome assemblies have discovered new sequences that are not in the current reference genome 12,[14][15][16] . Systematic approach for discovering structural variations identifies many new structural variations and projects that we still needs more samples to generate a more comprehensive catalogs of larger variants in human population other than SNPs and small indels.…”

Section: Introductionmentioning

confidence: 99%

“…Nevertheless, with the fast pace in advance of computational methods and sequencing technologies, the cost to generate de novo human genome may drop to a price point that it has become affordable for personalized medicine soon. Our method will also help to build pan-human-genomics references which allows us to capture novel human genome sequences that are not available in the current human reference GRCh38 12,15 . Given the potential to provide more comprehensive view for human genomes, we are hoping, the whole genome assembly approach will provide important information for genetic diseases that can not be revealed easily with re-sequencing approach.…”

Section: Introductionmentioning

confidence: 99%

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Human Genome Assembly in 100 Minutes

Chin¹,

Khalak²

2019

Preprint

101

107

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De novo genome assembly provides comprehensive, unbiased genomic information and makes it possible to gain insight into new DNA sequences not present in reference genomes. Many de novo human genomes have been published in the last few years, leveraging a combination of inexpensive short-read and single-molecule long-read technologies. As long-read DNA sequencers become more prevalent, the computational burden of generating assemblies persists as a critical factor. The most common approach to long-read assembly, using an overlap-layout-consensus (OLC) paradigm, requires all-to-all read comparisons, which quadratically scales in computational complexity with the number of reads. We assert that recently achievements in sequencing technology (i.e. with accuracy ~99% and read length ~10-15k) enables a fundamentally better strategy for OLC that is effectively linear rather than quadratic. Our genome assembly implementation, Peregrine uses s parse hi erarchical m ini m iz er s (SHIMMER) to index reads thereby avoiding the need for an all-to-all read comparison step. Peregrine can assemble 30x human PacBio CCS read datasets in less than 30 CPU hours and around 100 wall-clock minutes to a high contiguity assembly (N50 > 20Mb). The continued advance of sequencing technologies coupled with the Peregrine assembler enables routine generation of human de novo assemblies. This will allow for population scale measurements of more comprehensive genomic variations --beyond SNPs and small indels -as well as novel applications requiring rapid access to de novo assemblies.

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De novoassembly of two Swedish genomes reveals missing segments from the human GRCh38 reference and improves variant calling of population-scale sequencing data

Abstract: We have performed de novo assembly of two Swedish genomes using long-read sequencing and optical mapping, resulting in total assembly sizes of nearly 3 Gb and

Cited by 14 publications

References 39 publications

Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

Accurate characterization of expanded tandem repeat length and sequence through whole genome long-read sequencing on PromethION

Human Genome Assembly in 100 Minutes

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