Cargando…
RNA sequencing read depth requirement for optimal transcriptome coverage in Hevea brasiliensis
BACKGROUND: One 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 bra...
Autores principales: | , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2014
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926681/ https://www.ncbi.nlm.nih.gov/pubmed/24484543 http://dx.doi.org/10.1186/1756-0500-7-69 |
_version_ | 1782304011281498112 |
---|---|
author | Chow, Keng-See Ghazali, Ahmad-Kamal Hoh, Chee-Choong Mohd-Zainuddin, Zainorlina |
author_facet | Chow, Keng-See Ghazali, Ahmad-Kamal Hoh, Chee-Choong Mohd-Zainuddin, Zainorlina |
author_sort | Chow, Keng-See |
collection | PubMed |
description | BACKGROUND: One 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. FINDINGS: We 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. CONCLUSIONS: We 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. |
format | Online Article Text |
id | pubmed-3926681 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-39266812014-02-18 RNA sequencing read depth requirement for optimal transcriptome coverage in Hevea brasiliensis Chow, Keng-See Ghazali, Ahmad-Kamal Hoh, Chee-Choong Mohd-Zainuddin, Zainorlina BMC Res Notes Technical Note BACKGROUND: One 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. FINDINGS: We 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. CONCLUSIONS: We 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. BioMed Central 2014-02-01 /pmc/articles/PMC3926681/ /pubmed/24484543 http://dx.doi.org/10.1186/1756-0500-7-69 Text en Copyright © 2014 Chow et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Technical Note Chow, Keng-See Ghazali, Ahmad-Kamal Hoh, Chee-Choong Mohd-Zainuddin, Zainorlina RNA sequencing read depth requirement for optimal transcriptome coverage in Hevea brasiliensis |
title | RNA sequencing read depth requirement for optimal transcriptome coverage in Hevea brasiliensis |
title_full | RNA sequencing read depth requirement for optimal transcriptome coverage in Hevea brasiliensis |
title_fullStr | RNA sequencing read depth requirement for optimal transcriptome coverage in Hevea brasiliensis |
title_full_unstemmed | RNA sequencing read depth requirement for optimal transcriptome coverage in Hevea brasiliensis |
title_short | RNA sequencing read depth requirement for optimal transcriptome coverage in Hevea brasiliensis |
title_sort | rna sequencing read depth requirement for optimal transcriptome coverage in hevea brasiliensis |
topic | Technical Note |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926681/ https://www.ncbi.nlm.nih.gov/pubmed/24484543 http://dx.doi.org/10.1186/1756-0500-7-69 |
work_keys_str_mv | AT chowkengsee rnasequencingreaddepthrequirementforoptimaltranscriptomecoverageinheveabrasiliensis AT ghazaliahmadkamal rnasequencingreaddepthrequirementforoptimaltranscriptomecoverageinheveabrasiliensis AT hohcheechoong rnasequencingreaddepthrequirementforoptimaltranscriptomecoverageinheveabrasiliensis AT mohdzainuddinzainorlina rnasequencingreaddepthrequirementforoptimaltranscriptomecoverageinheveabrasiliensis |