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Resolving the full spectrum of human genome variation using Linked-Reads.

ABSTRACT: Large-scale population analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short-read whole-genome sequencing. However, these short-read approaches fail to give a complete picture of a genome. They struggle to identify structural events, cannot access repetitive regions, and fail to resolve the human genome into haplotypes. Here, we describe an approach that retains long range information while maintaining the advantages of short reads. Starting from ∼1 ng of high molecular weight DNA, we produce barcoded short-read libraries. Novel informatic approaches allow for the barcoded short reads to be associated with their original long molecules producing a novel data type known as "Linked-Reads". This approach allows for simultaneous detection of small and large variants from a single library. In this manuscript, we show the advantages of Linked-Reads over standard short-read approaches for reference-based analysis. Linked-Reads allow mapping to 38 Mb of sequence not accessible to short reads, adding sequence in 423 difficult-to-sequence genes including disease-relevant genes STRC, SMN1, and SMN2 Both Linked-Read whole-genome and whole-exome sequencing identify complex structural variations, including balanced events and single exon deletions and duplications. Further, Linked-Reads extend the region of high-confidence calls by 68.9 Mb. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

SUBMITTER: Marks P

PROVIDER: S-EPMC6442396 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Resolving the Full Spectrum of Human Genome Variation using Linked-Reads

Project description:Large-scale population based analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. Standard short-read approaches, used primarily due to accuracy, throughput and costs, fail to give a complete picture of a genome. They struggle to identify large, balanced structural events, cannot access repetitive regions of the genome and fail to resolve the human genome into its two haplotypes. Here we describe an approach that retains long range information while harnessing the power of short reads. Starting from only 1ng of DNA, we produce barcoded short read libraries. The use of novel informatic approaches allows for the barcoded short reads to be associated with the long molecules of origin producing a novel datatype known as 'Linked-Reads'. This approach allows for simultaneous detection of small and large variants from a single Linked-Read library. We have previously demonstrated the utility of whole genome Linked-Reads (lrWGS) for performing diploid, de novo assembly of individual genomes. In this manuscript, we show the utility of reference based analysis using a single Linked-Read library for full spectrum genome analysis. We demonstrate the ability of Linked-Reads to reconstruct megabase scale haplotypes and to recover parts of the genome that are typically inaccessible to short reads, including phenotypically important genes such as STRC, SMN1 and SMN2. We demonstrate the ability of both lrWGS and Linked-Read Whole Exome Sequencing (lrWES) to identify complex structural variations, including balanced events, single exon deletions, and single exon duplications. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

| EGAS00001003121 | EGA

Resolving the Full Spectrum of Human Genome Variation using Linked-Reads

Project description:Standard short-read approaches struggle to identify large, balanced structural events, cannot access repetitive regions of the genome and fail to resolve the human genome into its two haplotypes. Starting from only ~1ng of DNA, we produce barcoded short read libraries that retain long range information. We demonstrate the ability of both lrWGS and Linked-Read Whole Exome Sequencing (lrWES) to identify complex structural variations, including balanced events, single exon deletions, and single exon duplications. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

2018-08-21 | PRJEB28297 | EVA

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

Project description:BackgroundProducing 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.ResultsWe 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.ConclusionsThe 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.

| S-EPMC6879002 | biostudies-literature

Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads.

Project description:The Illumina DNA sequencing platform generates accurate but short reads, which can be used to produce accurate but fragmented genome assemblies. Pacific Biosciences and Oxford Nanopore Technologies DNA sequencing platforms generate long reads that can produce complete genome assemblies, but the sequencing is more expensive and error-prone. There is significant interest in combining data from these complementary sequencing technologies to generate more accurate "hybrid" assemblies. However, few tools exist that truly leverage the benefits of both types of data, namely the accuracy of short reads and the structural resolving power of long reads. Here we present Unicycler, a new tool for assembling bacterial genomes from a combination of short and long reads, which produces assemblies that are accurate, complete and cost-effective. Unicycler builds an initial assembly graph from short reads using the de novo assembler SPAdes and then simplifies the graph using information from short and long reads. Unicycler uses a novel semi-global aligner to align long reads to the assembly graph. Tests on both synthetic and real reads show Unicycler can assemble larger contigs with fewer misassemblies than other hybrid assemblers, even when long-read depth and accuracy are low. Unicycler is open source (GPLv3) and available at github.com/rrwick/Unicycler.

| S-EPMC5481147 | biostudies-literature

ARCS: scaffolding genome drafts with linked reads.

Project description:MotivationSequencing of human genomes is now routine, and assembly of shotgun reads is increasingly feasible. However, assemblies often fail to inform about chromosome-scale structure due to a lack of linkage information over long stretches of DNA-a shortcoming that is being addressed by new sequencing protocols, such as the GemCode and Chromium linked reads from 10 × Genomics.ResultsHere, we present ARCS, an application that utilizes the barcoding information contained in linked reads to further organize draft genomes into highly contiguous assemblies. We show how the contiguity of an ABySS H.sapiens genome assembly can be increased over six-fold, using moderate coverage (25-fold) Chromium data. We expect ARCS to have broad utility in harnessing the barcoding information contained in linked read data for connecting high-quality sequences in genome assembly drafts.Availability and implementationhttps://github.com/bcgsc/ARCS/.Contactrwarren@bcgsc.ca.Supplementary informationSupplementary data are available at Bioinformatics online.

| S-EPMC6030987 | biostudies-literature

Using long and linked reads to improve an Atlantic herring (Clupea harengus) genome assembly.

Project description:Atlantic herring (Clupea harengus) is one of the most abundant fish species in the world. It is an important economical and nutritional resource, as well as a crucial part of the North Atlantic ecosystem. In 2016, a draft herring genome assembly was published. Being a species of such importance, we sought to independently verify and potentially improve the herring genome assembly. We sequenced the herring genome generating paired-end, mate-pair, linked and long reads. Three assembly versions of the herring genome were generated based on a de novo assembly (A1), which was scaffolded using linked and long reads (A2) and then merged with the previously published assembly (A3). The resulting assemblies were compared using parameters describing the size, fragmentation, correctness, and completeness of the assemblies. Results showed that the A2 assembly was less fragmented, more complete and more correct than A1. A3 showed improvement in fragmentation and correctness compared with A2 and the published assembly but was slightly less complete than the published assembly. Thus, we here confirmed the previously published herring assembly, and made improvements by further scaffolding the assembly and removing low-quality sequences using linked and long reads and merging of assemblies.

| S-EPMC6881392 | biostudies-literature

Genome Assembly of the A-Group Wolbachia in Nasonia oneida Using Linked-Reads Technology.

Project description:Wolbachia are obligate intracellular bacteria which commonly infect various nematode and arthropod species. Genome sequences have been generated from arthropod samples following enrichment for the intracellular bacteria, and genomes have also been assembled from arthropod whole-genome sequencing projects. However, these methods remain challenging for infections that occur at low titers in hosts. Here we report the first Wolbachia genome assembled from host sequences using 10× Genomics linked-reads technology. The high read depth attainable by this method allows for recovery of intracellular bacteria that are at low concentrations. Based on the depth differences (714× for the insect and 59× for the bacterium), we assembled the genome of a Wolbachia in the parasitoid jewel wasp species Nasonia oneida. The final draft assembly consists of 1,293, 06 bp in 47 scaffolds with 1,114 coding genes and 97.01% genome completeness assessed by checkM. Comparisons of the five Multi Locus Sequence Typing genes revealed that the sequenced Wolbachia genome is the A1 strain (henceforth wOneA1) previously reported in N. oneida. Pyrosequencing confirms that the wasp strain lacks A2 and B types previously detected in this insect, which were likely lost during laboratory culturing. Assembling bacterial genomes from host genome projects can provide an effective method for sequencing bacterial genomes, even when the infections occur at low density in sampled tissues.

| S-EPMC6821208 | biostudies-literature

Cost-effective assembly of the African wild dog (Lycaon pictus) genome using linked reads.

Project description:A high-quality reference genome assembly is a valuable tool for the study of non-model organisms. Genomic techniques can provide important insights about past population sizes and local adaptation and can aid in the development of breeding management plans. This information is important for fields such as conservation genetics, where endangered species require critical and immediate attention. However, funding for genomic-based methods can be sparse for conservation projects, as costs for general species management can consume budgets. Here, we report the generation of high-quality reference genomes for the African wild dog (Lycaon pictus) at a low cost (<$3000), thereby facilitating future studies of this endangered canid. We generated assemblies for three individuals using the linked-read 10x Genomics Chromium system. The most continuous assembly had a scaffold and contig N50 of 21 Mb and 83 Kb, respectively, and completely reconstructed 95% of a set of conserved mammalian genes. Additionally, we estimate the heterozygosity and demographic history of African wild dogs, revealing that although they have historically low effective population sizes, heterozygosity remains high. We show that 10x Genomics Chromium data can be used to effectively generate high-quality genomes from Illumina short-read data of intermediate coverage (∼25x-50x). Interestingly, the wild dog shows higher heterozygosity than other species of conservation concern, possibly due to its behavioral ecology. The availability of reference genomes for non-model organisms will facilitate better genetic monitoring of threatened species such as the African wild dog and help conservationists to better understand the ecology and adaptability of those species in a changing environment.

| S-EPMC6350039 | biostudies-literature

Resolving repeat families with long reads.

Project description:BackgroundDraft quality genomes for a multitude of organisms have become common due to the advancement of genome assemblers using long-read technologies with high error rates. Although current assemblies are substantially more contiguous than assemblies based on short reads, complete chromosomal assemblies are still challenging. Interspersed repeat families with multiple copy versions dominate the contig and scaffold ends of current long-read assemblies for complex genomes. These repeat families generally remain unresolved, as existing algorithmic solutions either do not scale to large copy numbers or can not handle the current high read error rates.ResultsWe propose novel repeat resolution methods for large interspersed repeat families and assess their accuracy on simulated data sets with various distinct repeat structures and on drosophila melanogaster transposons. Additionally, we compare our methods to an existing long read repeat resolution tool and show the improved accuracy of our method.ConclusionsOur results demonstrate the applicability of our methods for the improvement of the contiguity of genome assemblies.

| S-EPMC6506941 | biostudies-literature

REXTAL: Regional Extension of Assemblies Using Linked-Reads.

Project description:It is currently impossible to get complete de-novo assembly of segmentally duplicated genome regions using genome-wide short-read datasets. Here, we devise a new computational method called Regional Extension of Assemblies Using Linked-Reads (REXTAL) for improved region-specific assembly of segmental duplication-containing DNA, leveraging genomic short-read datasets generated from large DNA molecules partitioned and barcoded using the "Gel Bead in Emulsion" (GEM) microfluidic method (Zheng et al., 2016). We show that using REXTAL, it is possible to extend assembly of single-copy diploid DNA into adjacent, otherwise inaccessible subtelomere segmental duplication regions and other subtelomeric gap regions. Moreover, REXTAL is computationally more efficient for the directed assembly of such regions from multiple genomes (e.g., for the comparison of structural variation) than genome-wide assembly approaches.

| S-EPMC6996091 | biostudies-literature

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