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Targeted short read sequencing and assembly of re-arrangements and candidate gene loci provide megabase diplotypes.

ABSTRACT: The human genome is composed of two haplotypes, otherwise called diplotypes, which denote phased polymorphisms and structural variations (SVs) that are derived from both parents. Diplotypes place genetic variants in the context of cis-related variants from a diploid genome. As a result, they provide valuable information about hereditary transmission, context of SV, regulation of gene expression and other features which are informative for understanding human genetics. Successful diplotyping with short read whole genome sequencing generally requires either a large population or parent-child trio samples. To overcome these limitations, we developed a targeted sequencing method for generating megabase (Mb)-scale haplotypes with short reads. One selects specific 0.1-0.2 Mb high molecular weight DNA targets with custom-designed Cas9-guide RNA complexes followed by sequencing with barcoded linked reads. To test this approach, we designed three assays, targeting the BRCA1 gene, the entire 4-Mb major histocompatibility complex locus and 18 well-characterized SVs, respectively. Using an integrated alignment- and assembly-based approach, we generated comprehensive variant diplotypes spanning the entirety of the targeted loci and characterized SVs with exact breakpoints. Our results were comparable in quality to long read sequencing.

SUBMITTER: Shin G

PROVIDER: S-EPMC6821272 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Targeted short read sequencing and assembly of re-arrangements and candidate gene loci provide megabase diplotypes.

Shin GiWon G Greer Stephanie U SU Xia Li C LC Lee HoJoon H Zhou Jun J Boles T Christian TC Ji Hanlee P HP

Nucleic acids research 20191101 19

The human genome is composed of two haplotypes, otherwise called diplotypes, which denote phased polymorphisms and structural variations (SVs) that are derived from both parents. Diplotypes place genetic variants in the context of cis-related variants from a diploid genome. As a result, they provide valuable information about hereditary transmission, context of SV, regulation of gene expression and other features which are informative for understanding human genetics. Successful diplotyping with ...[more]

PMID: 31350896

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Project description:BACKGROUND: RNA-seq has shown huge potential for phylogenomic inferences in non-model organisms. However, error, incompleteness, and redundant assembled transcripts for each gene in de novo assembly of short reads cause noise in analyses and a large amount of missing data in the aligned matrix. To address these problems, we compare de novo assemblies of paired end 90 bp RNA-seq reads using Oases, Trinity, Trans-ABySS and SOAPdenovo-Trans to transcripts from genome annotation of the model plant Ricinus communis. By doing so we evaluate strategies for optimizing total gene coverage and minimizing assembly chimeras and redundancy. RESULTS: We found that the frequency and structure of chimeras vary dramatically among different software packages. The differences were largely due to the number of trans-self chimeras that contain repeats in the opposite direction. More than half of the total chimeras in Oases and Trinity were trans-self chimeras. Within each package, we found a trade-off between maximizing reference coverage and minimizing redundancy and chimera rate. In order to reduce redundancy, we investigated three methods: 1) using cap3 and CD-HIT-EST to combine highly similar transcripts, 2) only retaining the transcript with the highest read coverage, or removing the transcript with the lowest read coverage for each subcomponent in Trinity, and 3) filtering Oases single k-mer assemblies by number of transcripts per locus and relative transcript length, and then finding the transcript with the highest read coverage. We then utilized results from blastx against model protein sequences to effectively remove trans chimeras. After optimization, seven assembly strategies among all four packages successfully assembled 42.9-47.1% of reference genes to more than 200 bp, with a chimera rate of 0.92-2.21%, and on average 1.8-3.1 transcripts per reference gene assembled. CONCLUSIONS: With rapidly improving sequencing and assembly tools, our study provides a framework to benchmark and optimize performance before choosing tools or parameter combinations for analyzing short-read RNA-seq data. Our study demonstrates that choice of assembly package, k-mer sizes, post-assembly redundancy-reduction and chimera cleanup, and strand-specific RNA-seq library preparation and assembly dramatically improves gene coverage by non-redundant and non-chimeric transcripts that are optimized for downstream phylogenomic analyses.

Dataset Information

Targeted short read sequencing and assembly of re-arrangements and candidate gene loci provide megabase diplotypes.

Publications

Targeted short read sequencing and assembly of re-arrangements and candidate gene loci provide megabase diplotypes.

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