scholarly journals How well can we create phased, diploid, human genomes?: An assessment of FALCON-Unzip phasing using a human trio

2018 ◽  
Author(s):  
Arkarachai Fungtammasan ◽  
Brett Hannigan
Keyword(s):  
De Novo ◽  
Personal Genome ◽  
Specific Expression ◽  
Human Genomes ◽  
Future Improvement ◽  
Long Read ◽  
Allele Specific ◽  
Personal Genomes ◽  
Reference Genomes ◽  
Haplotype Information

ABSTRACTLong read sequencing technology has allowed researchers to create de novo assemblies with impressive continuity[1,2]. This advancement has dramatically increased the number of reference genomes available and hints at the possibility of a future where personal genomes are assembled rather than resequenced. In 2016 Pacific Biosciences released the FALCON-Unzip framework, which can provide long, phased haplotype contigs from de novo assemblies. This phased genome algorithm enhances the accuracy of highly heterozygous organisms and allows researchers to explore questions that require haplotype information such as allele-specific expression and regulation. However, validation of this technique has been limited to small genomes or inbred individuals[3].As a roadmap to personal genome assembly and phasing, we assess the phasing accuracy of FALCON-Unzip in humans using publicly available data for the Ashkenazi trio from the Genome in a Bottle Consortium[4]. To assess the accuracy of the Unzip algorithm, we assembled the genome of the son using FALCON and FALCON Unzip, genotyped publicly available short read data for the mother and the father, and observed the inheritance pattern of the parental SNPs along the phased genome of the son. We found that 72.8% of haplotype contigs share SNPs with only one parent suggesting that these contigs are correctly phased. Most mis-phased SNPs are random but present in high frequency toward the end of haplotype contigs. Approximately 20.7% of mis-phased haplotype contigs contain clusters of mis-phased SNPs, suggesting that haplotypes were mis-joined by FALCON-Unzip. Mis-joined boundaries in those contigs are located in areas of low SNP density. This research demonstrates that the FALCON-Unzip algorithm can be used to create long and accurate haplotypes for humans and identifies problematic regions that could benefit in future improvement.

scholarly journals Comparative Annotation Toolkit (CAT) - simultaneous clade and personal genome annotation

2017 ◽  
Author(s):  
Ian T. Fiddes ◽  
Joel Armstrong ◽  
Mark Diekhans ◽  
Stefanie Nachtweide ◽  
Zev N. Kronenberg ◽  
...  
Keyword(s):  
Genome Annotation ◽  
De Novo ◽  
Low Cost ◽  
Great Apes ◽  
Personal Genome ◽  
Sequencing Technologies ◽  
Human Genomes ◽  
Long Read ◽  
Genome Assemblies ◽  
Rat Genome

ABSTRACTThe recent introductions of low-cost, long-read, and read-cloud sequencing technologies coupled with intense efforts to develop efficient algorithms have made affordable, high-quality de novo sequence assembly a realistic proposition. The result is an explosion of new, ultra-contiguous genome assemblies. To compare these genomes we need robust methods for genome annotation. We describe the fully open source Comparative Annotation Toolkit (CAT), which provides a flexible way to simultaneously annotate entire clades and identify orthology relationships. We show that CAT can be used to improve annotations on the rat genome, annotate the great apes, annotate a diverse set of mammals, and annotate personal, diploid human genomes. We demonstrate the resulting discovery of novel genes, isoforms and structural variants, even in genomes as well studied as rat and the great apes, and how these annotations improve cross-species RNA expression experiments.

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

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.

scholarly journals GeneiASE: Detection of condition-dependent and static allele-specific expression from RNA-seq data without haplotype information

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Daniel Edsgärd ◽  
Maria Jesus Iglesias ◽  
Sarah-Jayne Reilly ◽  
Anders Hamsten ◽  
Per Tornvall ◽  
...  
Keyword(s):  
Rna Seq ◽  
Specific Expression ◽  
Allele Specific Expression ◽  
Allele Specific ◽  
Haplotype Information

scholarly journals Merqury: reference-free quality, completeness, and phasing assessment for genome assemblies

2020 ◽  
Author(s):  
Arang Rhie ◽  
Brian P. Walenz ◽  
Sergey Koren ◽  
Adam M. Phillippy
Keyword(s):  
De Novo ◽  
High Accuracy ◽  
Link Type ◽  
Base Level ◽  
Project Home Page ◽  
Set Operations ◽  
Assembly Evaluation ◽  
Long Read ◽  
Genome Assemblies ◽  
Reference Genomes

AbstractRecent 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.Availability of data and materialProject name: MerquryProject home page: https://github.com/marbl/merqury, https://github.com/marbl/merylArchived version: https://github.com/marbl/merqury/releases/tag/v1.0Operating system(s): Platform independentProgramming language: C++, Java, PerlOther requirements: gcc 4.8 or higher, java 1.6 or higherLicense: Public domain (see https://github.com/marbl/merqury/blob/master/README.license) Any restrictions to use by non-academics: No restrictions applied

scholarly journals Transcriptome variation in human tissues revealed by long-read sequencing

2021 ◽  
Author(s):  
Dafni A Glinos ◽  
Garrett Garborcauskas ◽  
Paul Hoffman ◽  
Nava Ehsan ◽  
Lihua Jiang ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rare Variants ◽  
Similar Proportion ◽  
Specific Expression ◽  
Sequencing Technologies ◽  
Cellular Environment ◽  
Long Read ◽  
Allele Specific ◽  
Transcript Structure ◽  
Regulatory Effects

SummaryRegulation of transcript structure generates transcript diversity and plays an important role in human disease. The advent of long-read sequencing technologies offers the opportunity to study the role of genetic variation in transcript structure. In this paper, we present a large human long-read RNA-seq dataset using the Oxford Nanopore Technologies platform from 88 samples from GTEx tissues and cell lines, complementing the GTEx resource. We identified just under 100,000 new transcripts for annotated genes, and validated the protein expression of a similar proportion of novel and annotated transcripts. We developed a new computational package, LORALS, to analyze genetic effects of rare and common variants on the transcriptome via allele-specific analysis of long reads. We called allele-specific expression and transcript structure events, providing novel insights into the specific transcript alterations caused by common and rare genetic variants and highlighting the resolution gained from long-read data. We were able to perturb transcript structure upon knockdown of PTBP1, an RNA binding protein that mediates splicing, thereby finding genetic regulatory effects that are modified by the cellular environment. Finally, we use this dataset to enhance variant interpretation and study rare variants leading to aberrant splicing patterns.

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

2019 ◽  
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.

scholarly journals NextSV: a meta-caller for structural variants from low-coverage long-read sequencing data

2016 ◽  
Author(s):  
Li Fang ◽  
Jiang Hu ◽  
Depeng Wang ◽  
Kai Wang
Keyword(s):  
Whole Genome ◽  
Ashkenazi Jewish ◽  
Structural Variants ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing ◽  
Human Genomes ◽  
Long Read ◽  
Personal Genomes ◽  
Low Coverage

AbstractBackgroundStructural variants (SVs) in human genomes are implicated in a variety of human diseases. Long-read sequencing delivers much longer read lengths than short-read sequencing and may greatly improve SV detection. However, due to the relatively high cost of long-read sequencing, it is unclear what coverage is needed and how to optimally use the aligners and SV callers.ResultsIn this study, we developed NextSV, a meta-caller to perform SV calling from low coverage long-read sequencing data. NextSV integrates three aligners and three SV callers and generates two integrated call sets (sensitive/stringent) for different analysis purposes. We evaluated SV calling performance of NextSV under different PacBio coverages on two personal genomes, NA12878 and HX1. Our results showed that, compared with running any single SV caller, NextSV stringent call set had higher precision and balanced accuracy (F1 score) while NextSV sensitive call set had a higher recall. At 10X coverage, the recall of NextSV sensitive call set was 93.5% to 94.1% for deletions and 87.9% to 93.2% for insertions, indicating that ~10X coverage might be an optimal coverage to use in practice, considering the balance between the sequencing costs and the recall rates. We further evaluated the Mendelian errors on an Ashkenazi Jewish trio dataset.ConclusionsOur results provide useful guidelines for SV detection from low coverage whole-genome PacBio data and we expect that NextSV will facilitate the analysis of SVs on long-read sequencing data.

scholarly journals Efficient de novo assembly of eleven human genomes using PromethION sequencing and a novel nanopore toolkit

2019 ◽  
Author(s):  
Kishwar Shafin ◽  
Trevor Pesout ◽  
Ryan Lorig-Roach ◽  
Marina Haukness ◽  
Hugh E. Olsen ◽  
...  
Keyword(s):  
Human Genome ◽  
De Novo ◽  
Proximity Ligation ◽  
Current State ◽  
Human Genomes ◽  
Sequencing Method ◽  
Human Genome Assembly ◽  
Long Read ◽  
Genome Assemblies ◽  
Assembly Performance

AbstractPresent workflows for producing human genome assemblies from long-read technologies have cost and production time bottlenecks that prohibit efficient scaling to large cohorts. We demonstrate an optimized PromethION nanopore sequencing method for eleven human genomes. The sequencing, performed on one machine in nine days, achieved an average 63x coverage, 42 Kb read N50, 90% median read identity and 6.5x coverage in 100 Kb+ reads using just three flow cells per sample. To assemble these data we introduce new computational tools: Shasta - a de novo long read assembler, and MarginPolish & HELEN - a suite of nanopore assembly polishing algorithms. On a single commercial compute node Shasta can produce a complete human genome assembly in under six hours, and MarginPolish & HELEN can polish the result in just over a day, achieving 99.9% identity (QV30) for haploid samples from nanopore reads alone. We evaluate assembly performance for diploid, haploid and trio-binned human samples in terms of accuracy, cost, and time and demonstrate improvements relative to current state-of-the-art methods in all areas. We further show that addition of proximity ligation (Hi-C) sequencing yields near chromosome-level scaffolds for all eleven genomes.

scholarly journals Extensive sequencing of seven human genomes to characterize benchmark reference materials

2015 ◽  
Author(s):  
Justin M Zook ◽  
David Catoe ◽  
Jennifer McDaniel ◽  
Lindsay Vang ◽  
Noah Spies ◽  
...  
Keyword(s):  
Human Genome ◽  
Reference Materials ◽  
De Novo ◽  
Variant Calling ◽  
Genome Project ◽  
Genome Comparison ◽  
Personal Genome ◽  
Sequencing Data ◽  
Sequencing Technologies ◽  
Human Genomes

The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCodeTM WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly.

scholarly journals Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads

2019 ◽  
Author(s):  
Mitchell R. Vollger ◽  
Glennis A. Logsdon ◽  
Peter A. Audano ◽  
Arvis Sulovari ◽  
David Porubsky ◽  
...  
Keyword(s):  
Human Genome ◽  
Single Molecule ◽  
Tandem Repeats ◽  
De Novo ◽  
Sequence Data ◽  
Gene Annotation ◽  
Hydatidiform Mole ◽  
High Fidelity ◽  
Human Genomes ◽  
Long Read

AbstractThe sequence and assembly of human genomes using long-read sequencing technologies has revolutionized our understanding of structural variation and genome organization. We compared the accuracy, continuity, and gene annotation of genome assemblies generated from either high-fidelity (HiFi) or continuous long-read (CLR) datasets from the same complete hydatidiform mole human genome. We find that the HiFi sequence data assemble an additional 10% of duplicated regions and more accurately represent the structure of tandem repeats, as validated with orthogonal analyses. As a result, an additional 5 Mbp of pericentromeric sequences are recovered in the HiFi assembly, resulting in a 2.5-fold increase in the NG50 within 1 Mbp of the centromere (HiFi 480.6 kbp, CLR 191.5 kbp). Additionally, the HiFi genome assembly was generated in significantly less time with fewer computational resources than the CLR assembly. Although the HiFi assembly has significantly improved continuity and accuracy in many complex regions of the genome, it still falls short of the assembly of centromeric DNA and the largest regions of segmental duplication using existing assemblers. Despite these shortcomings, our results suggest that HiFi may be the most effective stand-alone technology for de novo assembly of human genomes.

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