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Biases in read coverage demonstrated by interlaboratory and interplatform comparison of 117 mRNA and genome sequencing experiments.

ABSTRACT: High-throughput sequencing of whole genomes and transcriptomes allows one to generate large amounts of sequence data very rapidly and at a low cost. The goal of most mRNA sequencing studies is to perform the comparison of the expression level between different samples. However, given a broad variety of modern sequencing protocols, platforms and versions thereof, it is not clear to what extent the obtained results are consistent across platforms and laboratories. The comparison of 117 human mRNA and genome high-throughput sequencing experiments performed on the Illumina and SOLiD platforms at 26 institutions all over the world demonstrated high dependency of the gene coverage profiles on the producing laboratory. Gene coverage profiles showed laboratory-specific non-uniformity that survived the 3'-bias correction and mappability normalization, suggesting that there are other yet unknown mRNA-associated biases.

SUBMITTER: Khrameeva EE

PROVIDER: S-EPMC3358657 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Biases in read coverage demonstrated by interlaboratory and interplatform comparison of 117 mRNA and genome sequencing experiments.

Khrameeva Ekaterina E EE Gelfand Mikhail S MS

BMC bioinformatics 20120419

High-throughput sequencing of whole genomes and transcriptomes allows one to generate large amounts of sequence data very rapidly and at a low cost. The goal of most mRNA sequencing studies is to perform the comparison of the expression level between different samples. However, given a broad variety of modern sequencing protocols, platforms and versions thereof, it is not clear to what extent the obtained results are consistent across platforms and laboratories. The comparison of 117 human mRNA ...[more]

PMID: 22537043

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Project description:BackgroundOne of the concerns of assembling de novo transcriptomes is determining the amount of read sequences required to ensure a comprehensive coverage of genes expressed in a particular sample. In this report, we describe the use of Illumina paired-end RNA-Seq (PE RNA-Seq) reads from Hevea brasiliensis (rubber tree) bark to devise a transcript mapping approach for the estimation of the read amount needed for deep transcriptome coverage.FindingsWe optimized the assembly of a Hevea bark transcriptome based on 16 Gb Illumina PE RNA-Seq reads using the Oases assembler across a range of k-mer sizes. We then assessed assembly quality based on transcript N50 length and transcript mapping statistics in relation to (a) known Hevea cDNAs with complete open reading frames, (b) a set of core eukaryotic genes and (c) Hevea genome scaffolds. This was followed by a systematic transcript mapping process where sub-assemblies from a series of incremental amounts of bark transcripts were aligned to transcripts from the entire bark transcriptome assembly. The exercise served to relate read amounts to the degree of transcript mapping level, the latter being an indicator of the coverage of gene transcripts expressed in the sample. As read amounts or datasize increased toward 16 Gb, the number of transcripts mapped to the entire bark assembly approached saturation. A colour matrix was subsequently generated to illustrate sequencing depth requirement in relation to the degree of coverage of total sample transcripts.ConclusionsWe devised a procedure, the "transcript mapping saturation test", to estimate the amount of RNA-Seq reads needed for deep coverage of transcriptomes. For Hevea de novo assembly, we propose generating between 5-8 Gb reads, whereby around 90% transcript coverage could be achieved with optimized k-mers and transcript N50 length. The principle behind this methodology may also be applied to other non-model plants, or with reads from other second generation sequencing platforms.

Dataset Information

Biases in read coverage demonstrated by interlaboratory and interplatform comparison of 117 mRNA and genome sequencing experiments.

Publications

Biases in read coverage demonstrated by interlaboratory and interplatform comparison of 117 mRNA and genome sequencing experiments.

Similar Datasets

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