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Long fragments achieve lower base quality in Illumina paired-end sequencing.

ABSTRACT: Illumina's technology provides high quality reads of DNA fragments with error rates below 1/1000 per base. Sequencing runs typically generate millions of reads in which the vast majority of the reads has an average error rate below 1/1000. However, some paired-end sequencing data show the presence of a subpopulation of reads where the second read (R2) has lower average qualities. We show that the fragment length is a major driver of increased error rates in the R2 reads. Fragments above 500 nt tend to yield lower base qualities and higher error rates than shorter fragments. We use publicly available Illumina data to demonstrate that the fragment length dependency of the R2 read qualities exists in various library protocols, in different labs and using different sequencer models. Our finding extends the understanding of the Illumina read quality and has implications on error models for Illumina reads. It also sheds a light on the importance of controlling the fragment size during library preparation.

SUBMITTER: Tan G

PROVIDER: S-EPMC6393434 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Long fragments achieve lower base quality in Illumina paired-end sequencing.

Tan Ge G Opitz Lennart L Schlapbach Ralph R Rehrauer Hubert H

Scientific reports 20190227 1

Illumina's technology provides high quality reads of DNA fragments with error rates below 1/1000 per base. Sequencing runs typically generate millions of reads in which the vast majority of the reads has an average error rate below 1/1000. However, some paired-end sequencing data show the presence of a subpopulation of reads where the second read (R2) has lower average qualities. We show that the fragment length is a major driver of increased error rates in the R2 reads. Fragments above 500 nt t ...[more]

PMID: 30814542

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Project description:BackgroundHouttuynia cordata Thunb. is an important traditional medical herb in China and other Asian countries, with high medicinal and economic value. However, a lack of available genomic information has become a limitation for research on this species. Thus, we carried out high-throughput transcriptomic sequencing of H. cordata to generate an enormous transcriptome sequence dataset for gene discovery and molecular marker development.Principal findingsIllumina paired-end sequencing technology produced over 56 million sequencing reads from H. cordata mRNA. Subsequent de novo assembly yielded 63,954 unigenes, 39,982 (62.52%) and 26,122 (40.84%) of which had significant similarity to proteins in the NCBI nonredundant protein and Swiss-Prot databases (E-value <10(-5)), respectively. Of these annotated unigenes, 30,131 and 15,363 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. In addition, 24,434 (38.21%) unigenes were mapped onto 128 pathways using the KEGG pathway database and 17,964 (44.93%) unigenes showed homology to Vitis vinifera (Vitaceae) genes in BLASTx analysis. Furthermore, 4,800 cDNA SSRs were identified as potential molecular markers. Fifty primer pairs were randomly selected to detect polymorphism among 30 samples of H. cordata; 43 (86%) produced fragments of expected size, suggesting that the unigenes were suitable for specific primer design and of high quality, and the SSR marker could be widely used in marker-assisted selection and molecular breeding of H. cordata in the future.ConclusionsThis is the first application of Illumina paired-end sequencing technology to investigate the whole transcriptome of H. cordata and to assemble RNA-seq reads without a reference genome. These data should help researchers investigating the evolution and biological processes of this species. The SSR markers developed can be used for construction of high-resolution genetic linkage maps and for gene-based association analyses in H. cordata. This work will enable future functional genomic research and research into the distinctive active constituents of this genus.

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Long fragments achieve lower base quality in Illumina paired-end sequencing.

Publications

Long fragments achieve lower base quality in Illumina paired-end sequencing.

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