Assessment of human diploid genome assembly with 10x Linked-Reads data

Abstract Background Producing cost-effective haplotype-resolved personal genomes remains challenging. 10x Linked-Read sequencing, with its high base quality and long-range information, has been demonstrated to facilitate de novo assembly of human genomes and variant detection. In this study, we investigate in depth how the parameter space of 10x library preparation and sequencing affects assembly quality, on the basis of both simulated and real libraries. Results We prepared and sequenced eight 10x libraries with a diverse set of parameters from standard cell lines NA12878 and NA24385 and performed whole-genome assembly on the data. We also developed the simulator LRTK-SIM to follow the workflow of 10x data generation and produce realistic simulated Linked-Read data sets. We found that assembly quality could be improved by increasing the total sequencing coverage (C) and keeping physical coverage of DNA fragments (CF) or read coverage per fragment (CR) within broad ranges. The optimal physical coverage was between 332× and 823× and assembly quality worsened if it increased to >1,000× for a given C. Long DNA fragments could significantly extend phase blocks but decreased contig contiguity. The optimal length-weighted fragment length (W${\mu _{FL}}$) was ∼50–150 kb. When broadly optimal parameters were used for library preparation and sequencing, ∼80% of the genome was assembled in a diploid state. Conclusions The Linked-Read libraries we generated and the parameter space we identified provide theoretical considerations and practical guidelines for personal genome assemblies based on 10x Linked-Read sequencing.

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Assessment of human diploid genome assembly with 10x Linked-Reads data

10.1101/729608 ◽

2019 ◽

Cited By ~ 2

Author(s):

Lu Zhang ◽

Xin Zhou ◽

Ziming Weng ◽

Arend Sidow

Keyword(s):

Parameter Space ◽

Genome Assembly ◽

De Novo ◽

Cost Effective ◽

Personal Genome ◽

Dna Fragments ◽

Data Generation ◽

Library Preparation ◽

Assembly Quality ◽

Practical Guidelines

AbstractBackgroundProducing cost-effective haplotype-resolved personal genomes remains challenging. 10x Linked-Read sequencing, with its high base quality and long-range information, has been demonstrated to facilitate de novo assembly of human genomes and variant detection. In this study, we investigate in depth how the parameter space of 10x library preparation and sequencing affects assembly quality, on the basis of both simulated and real libraries.FindingsWe prepared and sequenced eight 10x libraries with a diverse set of parameters from standard cell lines NA12878 and NA24385 and performed whole genome assembly on the data. We also developed the simulator LRTK-SIM to follow the workflow of 10x data generation and produce realistic simulated Linked-Read data sets. We found that assembly quality could be improved by increasing the total sequencing coverage (C) and keeping physical coverage of DNA fragments (CF) or read coverage per fragment (CR) within broad ranges. The optimal physical coverage was between 332X and 823X and assembly quality worsened if it increased to greater than 1,000X for a given C. Long DNA fragments could significantly extend phase blocks, but decreased contig contiguity. The optimal length-weighted fragment length (WμFL) was around 50 – 150kb. When broadly optimal parameters were used for library preparation and sequencing, ca. 80% of the genome was assembled in a diploid state.ConclusionThe Linked-Read libraries we generated and the parameter space we identified provide theoretical considerations and practical guidelines for personal genome assemblies based on 10x Linked-Read sequencing.

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A study of transposable element-associated structural variations (TASVs) using a de novo-assembled Korean genome

Experimental & Molecular Medicine ◽

10.1038/s12276-021-00586-y ◽

2021 ◽

Author(s):

Seyoung Mun ◽

Songmi Kim ◽

Wooseok Lee ◽

Keunsoo Kang ◽

Thomas J. Meyer ◽

...

Keyword(s):

Genome Sequencing ◽

Genome Assembly ◽

De Novo ◽

Personal Genome ◽

Human Populations ◽

Whole Genome ◽

Structural Variations ◽

Insert Size ◽

Human Genomes ◽

Next Generation Sequencing Ngs

AbstractAdvances in next-generation sequencing (NGS) technology have made personal genome sequencing possible, and indeed, many individual human genomes have now been sequenced. Comparisons of these individual genomes have revealed substantial genomic differences between human populations as well as between individuals from closely related ethnic groups. Transposable elements (TEs) are known to be one of the major sources of these variations and act through various mechanisms, including de novo insertion, insertion-mediated deletion, and TE–TE recombination-mediated deletion. In this study, we carried out de novo whole-genome sequencing of one Korean individual (KPGP9) via multiple insert-size libraries. The de novo whole-genome assembly resulted in 31,305 scaffolds with a scaffold N50 size of 13.23 Mb. Furthermore, through computational data analysis and experimental verification, we revealed that 182 TE-associated structural variation (TASV) insertions and 89 TASV deletions contributed 64,232 bp in sequence gain and 82,772 bp in sequence loss, respectively, in the KPGP9 genome relative to the hg19 reference genome. We also verified structural differences associated with TASVs by comparative analysis with TASVs in recent genomes (AK1 and TCGA genomes) and reported their details. Here, we constructed a new Korean de novo whole-genome assembly and provide the first study, to our knowledge, focused on the identification of TASVs in an individual Korean genome. Our findings again highlight the role of TEs as a major driver of structural variations in human individual genomes.

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LRSim: a Linked Reads Simulator generating insights for better genome partitioning

10.1101/103549 ◽

2017 ◽

Cited By ~ 1

Author(s):

Ruibang Luo ◽

Fritz J. Sedlazeck ◽

Charlotte A. Darby ◽

Stephen M. Kelly ◽

Michael C. Schatz

Keyword(s):

Genome Assembly ◽

Fragment Length ◽

De Novo ◽

Library Preparation ◽

Haplotype Phasing ◽

Multiple Datasets ◽

Long Read ◽

Fine Control ◽

Informatics Tools ◽

Sequencing Process

AbstractMotivationLinked reads are a form of DNA sequencing commercialized by 10X Genomics that uses highly multiplexed barcoding within microdroplets to tag short reads to progenitor molecules. The linked reads, spanning tens to hundreds of kilobases, offer an alternative to long-read sequencing for de novo assembly, haplotype phasing and other applications. However, there is no available simulator, making it difficult to measure their capability or develop new informatics tools.ResultsOur analysis of 13 real linked read datasets revealed their characteristics of barcodes, molecules and partitions. Based on this, we introduce LRSim that simulates linked reads by emulating the library preparation and sequencing process with fine control of 1) the number of simulated variants; 2) the linked-read characteristics; and 3) the Illumina reads profile. We conclude from the phasing and genome assembly of multiple datasets, recommendations on coverage, fragment length, and partitioning when sequencing human and non-human genome.AvailabilityLRSIM is under MIT license and is freely available at https://github.com/aquaskyline/[email protected]

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Aquila: diploid personal genome assembly and comprehensive variant detection based on linked reads

10.1101/660605 ◽

2019 ◽

Cited By ~ 1

Author(s):

Xin Zhou ◽

Lu Zhang ◽

Ziming Weng ◽

David L. Dill ◽

Arend Sidow

Keyword(s):

Genetic Variation ◽

Genome Sequence ◽

Genome Assembly ◽

Sequence Data ◽

Association Studies ◽

Cost Effective ◽

Whole Genome Sequence ◽

Personal Genome ◽

Whole Genome ◽

Nucleotide Polymorphisms

AbstractVariant discovery in personal, whole genome sequence data is critical for uncovering the genetic contributions to health and disease. We introduce a new approach, Aquila, that uses linked-read data for generating a high quality diploid genome assembly, from which it then comprehensively detects and phases personal genetic variation. Assemblies cover >95% of the human reference genome, with over 98% in a diploid state. Thus, the assemblies support detection and accurate genotyping of the most prevalent types of human genetic variation, including single nucleotide polymorphisms (SNPs), small insertions and deletions (small indels), and structural variants (SVs), in all but the most difficult regions. All heterozygous variants are phased in blocks that can approach arm-level length. The final output of Aquila is a diploid and phased personal genome sequence, and a phased VCF file that also contains homozygous and a few unphased heterozygous variants. Aquila represents a cost-effective evolution of whole-genome reconstruction that can be applied to cohorts for variation discovery or association studies, or to single individuals with rare phenotypes that could be caused by SVs or compound heterozygosity.

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De novo diploid genome assembly for genome-wide structural variant detection

10.1101/552430 ◽

2019 ◽

Cited By ~ 2

Author(s):

Lu Zhang ◽

Xin Zhou ◽

Ziming Weng ◽

Arend Sidow

Keyword(s):

De Novo ◽

Pairwise Alignment ◽

Cost Effective ◽

Personal Genome ◽

Ancestral State ◽

Short Fragment ◽

Fundamental Limitations ◽

Long Read ◽

Alternative Means ◽

Variant Detection

AbstractStructural variants (SVs) in a personal genome are important but, for all practical purposes, impossible to detect comprehensively by standard short-fragment sequencing. De novo assembly, traditionally used to generate reference genomes, offers an alternative means for variant detection and phasing but has not been applied broadly to human genomes because of fundamental limitations of short-fragment approaches and high cost of long-read technologies. We here show that 10x linked-read sequencing, which has been applied to assemble human diploid genomes into high quality contigs, supports accurate SV detection. We examined variants in six de novo 10x assemblies with diverse experimental parameters from two commonly used human cell lines, NA12878 and NA24385. The assemblies are effective in detecting mid-size SVs, which were discovered by simple pairwise alignment of the assemblies’ contigs to the reference (hg38). Our study also shows that the accuracy of SV breakpoint at base-pair level is high, with a majority (80% for deletion and 70% for insertion) of SVs having precisely correct sizes and breakpoints (<2bp difference). Finally, setting the ancestral state of SV loci by comparing to ape orthologs allows inference of the actual molecular mechanism (insertion or deletion) causing the mutation, which in about half of cases is opposite to that of the reference-based call. Interestingly, we uncover 214 SVs that may have been maintained as polymorphisms in the human lineage since before our divergence from chimp. Overall, we show that de novo assembly of 10x linked-read data can achieve cost-effective SV detection for personal genomes.

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Efficient long single molecule sequencing for cost effective and accurate sequencing, haplotyping, and de novo assembly

10.1101/324392 ◽

2018 ◽

Author(s):

Ou Wang ◽

Robert Chin ◽

Xiaofang Cheng ◽

Michelle Ka Wu ◽

Qing Mao ◽

...

Keyword(s):

Single Molecule ◽

Genome Assembly ◽

De Novo ◽

Low Cost ◽

Variant Calling ◽

Cost Effective ◽

High Quality ◽

Single Molecule Sequencing ◽

Single Tube ◽

Complex Structural

Obtaining accurate sequences from long DNA molecules is very important for genome assembly and other applications. Here we describe single tube long fragment read (stLFR), a technology that enables this a low cost. It is based on adding the same barcode sequence to sub-fragments of the original long DNA molecule (DNA co-barcoding). To achieve this efficiently, stLFR uses the surface of microbeads to create millions of miniaturized barcoding reactions in a single tube. Using a combinatorial process up to 3.6 billion unique barcode sequences were generated on beads, enabling practically non-redundant co-barcoding with 50 million barcodes per sample. Using stLFR, we demonstrate efficient unique co-barcoding of over 8 million 20-300 kb genomic DNA fragments. Analysis of the genome of the human genome NA12878 with stLFR demonstrated high quality variant calling and phasing into contigs up to N50 34 Mb. We also demonstrate detection of complex structural variants and complete diploid de novo assembly of NA12878. These analyses were all performed using single stLFR libraries and their construction did not significantly add to the time or cost of whole genome sequencing (WGS) library preparation. stLFR represents an easily automatable solution that enables high quality sequencing, phasing, SV detection, scaffolding, cost-effective diploid de novo genome assembly, and other long DNA sequencing applications.

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W2RAP: a pipeline for high quality, robust assemblies of large complex genomes from short read data

10.1101/110999 ◽

2017 ◽

Cited By ~ 9

Author(s):

Bernardo J. Clavijo ◽

Gonzalo Garcia Accinelli ◽

Jonathan Wright ◽

Darren Heavens ◽

Katie Barr ◽

...

Keyword(s):

De Novo ◽

Low Cost ◽

Cost Effective ◽

Data Generation ◽

Sequencing Data ◽

High Quality ◽

Crop Species ◽

Short Read ◽

Link Type ◽

Sequencing Technologies

AbstractProducing high-quality whole-genome shotgun de novo assemblies from plant and animal species with large and complex genomes using low-cost short read sequencing technologies remains a challenge. But when the right sequencing data, with appropriate quality control, is assembled using approaches focused on robustness of the process rather than maximization of a single metric such as the usual contiguity estimators, good quality assemblies with informative value for comparative analyses can be produced. Here we present a complete method described from data generation and qc all the way up to scaffold of complex genomes using Illumina short reads and its application to data from plants and human datasets. We show how to use the w2rap pipeline following a metric-guided approach to produce cost-effective assemblies. The assemblies are highly accurate, provide good coverage of the genome and show good short range contiguity. Our pipeline has already enabled the rapid, cost-effective generation of de novo genome assemblies from large, polyploid crop species with a focus on comparative genomics.Availabilityw2rap is available under MIT license, with some subcomponents under GPL-licenses. A ready-to-run docker with all software pre-requisites and example data is also available.http://github.com/bioinfologics/w2raphttp://github.com/bioinfologics/w2rap-contigger

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Chromosome-scale assembly comparison of the Korean Reference Genome KOREF from PromethION and PacBio with Hi-C mapping information

GigaScience ◽

10.1093/gigascience/giz125 ◽

2019 ◽

Vol 8 (12) ◽

Cited By ~ 6

Author(s):

Hui-Su Kim ◽

Sungwon Jeon ◽

Changjae Kim ◽

Yeon Kyung Kim ◽

Yun Sung Cho ◽

...

Keyword(s):

Human Genome ◽

Single Molecule ◽

Genome Assembly ◽

Reference Genome ◽

De Novo ◽

Low Cost ◽

Cost Effective ◽

Sequencing Data ◽

Smrt Sequencing ◽

Human Genome Assembly

Abstract Background Long DNA reads produced by single-molecule and pore-based sequencers are more suitable for assembly and structural variation discovery than short-read DNA fragments. For de novo assembly, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) are the favorite options. However, PacBio's SMRT sequencing is expensive for a full human genome assembly and costs more than $40,000 US for 30× coverage as of 2019. ONT PromethION sequencing, on the other hand, is 1/12 the price of PacBio for the same coverage. This study aimed to compare the cost-effectiveness of ONT PromethION and PacBio's SMRT sequencing in relation to the quality. Findings We performed whole-genome de novo assemblies and comparison to construct an improved version of KOREF, the Korean reference genome, using sequencing data produced by PromethION and PacBio. With PromethION, an assembly using sequenced reads with 64× coverage (193 Gb, 3 flowcell sequencing) resulted in 3,725 contigs with N50s of 16.7 Mb and a total genome length of 2.8 Gb. It was comparable to a KOREF assembly constructed using PacBio at 62× coverage (188 Gb, 2,695 contigs, and N50s of 17.9 Mb). When we applied Hi-C–derived long-range mapping data, an even higher quality assembly for the 64× coverage was achieved, resulting in 3,179 scaffolds with an N50 of 56.4 Mb. Conclusion The pore-based PromethION approach provided a high-quality chromosome-scale human genome assembly at a low cost with long maximum contig and scaffold lengths and was more cost-effective than PacBio at comparable quality measurements.

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Chromosome-scale assembly comparison of the Korean Reference Genome KOREF from PromethION and PacBio with Hi-C mapping information

10.1101/674804 ◽

2019 ◽

Cited By ~ 2

Author(s):

Hui-Su Kim ◽

Sungwon Jeon ◽

Changjae Kim ◽

Yeon Kyung Kim ◽

Yun Sung Cho ◽

...

Keyword(s):

Human Genome ◽

Single Molecule ◽

Genome Assembly ◽

Reference Genome ◽

De Novo ◽

Low Cost ◽

Cost Effective ◽

Sequencing Data ◽

Smrt Sequencing ◽

Human Genome Assembly

AbstractBackgroundLong DNA reads produced by single molecule and pore-based sequencers are more suitable for assembly and structural variation discovery than short read DNA fragments. For de novo assembly, PacBio and Oxford Nanopore Technologies (ONT) are favorite options. However, PacBio’s SMRT sequencing is expensive for a full human genome assembly and costs over 40,000 USD for 30x coverage as of 2019. ONT PromethION sequencing, on the other hand, is one-twelfth the price of PacBio for the same coverage. This study aimed to compare the cost-effectiveness of ONT PromethION and PacBio’s SMRT sequencing in relation to the quality.FindingsWe performed whole genome de novo assemblies and comparison to construct an improved version of KOREF, the Korean reference genome, using sequencing data produced by PromethION and PacBio. With PromethION, an assembly using sequenced reads with 64x coverage (193 Gb, 3 flowcell sequencing) resulted in 3,725 contigs with N50s of 16.7 Mbp and a total genome length of 2.8 Gbp. It was comparable to a KOREF assembly constructed using PacBio at 62x coverage (188 Gbp, 2,695 contigs and N50s of 17.9 Mbp). When we applied Hi-C-derived long-range mapping data, an even higher quality assembly for the 64x coverage was achieved, resulting in 3,179 scaffolds with an N50 of 56.4 Mbp.ConclusionThe pore-based PromethION approach provides a good quality chromosome-scale human genome assembly at a low cost with long maximum contig and scaffold lengths and is more cost-effective than PacBio at comparable quality measurements.

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Improved de novo Genome Assembly: Linked-Read Sequencing Combined with Optical Mapping Produce a High Quality Mammalian Genome at Relatively Low Cost

10.1101/128348 ◽

2017 ◽

Cited By ~ 10

Author(s):

DW Mohr ◽

A Naguib ◽

NI Weisenfeld ◽

V Kumar ◽

P Shah ◽

...

Keyword(s):

Genome Assembly ◽

De Novo ◽

Low Cost ◽

Cost Effective ◽

Conserved Synteny ◽

High Quality ◽

De Novo Genome Assembly ◽

Optical Maps ◽

Commercial Applications ◽

Hybrid Scaffolds

AbstractCurrent short-read methods have come to dominate genome sequencing because they are cost-effective, rapid, and accurate. However, short reads are most applicable when data can be aligned to a known reference. Two new methods for de novo assembly are linked-reads and restriction-site labeled optical maps. We combined commercial applications of these technologies for genome assembly of an endangered mammal, the Hawaiian Monk seal.We show that the linked-reads produced with 10X Genomics Chromium chemistry and assembled with Supernova v1.1 software produced scaffolds with an N50 of 22.23 Mbp with the longest individual scaffold of 84.06 Mbp. When combined with Bionano Genomics optical maps using Bionano RefAligner, the scaffold N50 increased to 29.65 Mbp for a total of 170 hybrid scaffolds, the longest of which was 84.78 Mbp. These results were 161X and 215X, respectively, improved over DISCOVAR de novo assemblies. The quality of the scaffolds was assessed using conserved synteny analysis of both the DNA sequence and predicted seal proteins relative to the genomes of humans and other species. We found large blocks of conserved synteny suggesting that the hybrid scaffolds were high quality. An inversion in one scaffold complementary to human chromosome 6 was found and confirmed by optical maps.The complementarity of linked-reads and optical maps is likely to make the production of high quality genomes more routine and economical and, by doing so, significantly improve our understanding of comparative genome biology.

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