Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage.
Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage.
复制标题
DOI：
10.1186/1471-2164-15-401
复制
发表时间：
2014-05-26
期刊：
影响因子：
4.4
通讯作者：
Preiss T
中科院分区：
文献类型：
--
作者：
Archer SK;Shirokikh NE;Preiss T
通过深度测序技术进行转录组分析的一个主要障碍是，丰富的转录本，如rRNA，可能会淹没文库，严重减少转录组的覆盖范围。去除这种不需要的序列的方法通常需要在文库制备之前对RNA样本进行处理，成本很高，不适合不寻常的物种和应用。在这里，我们描述了探针定向降解(PDD)，这是一种在单链cDNA文库阶段与DNA寡核苷酸杂交并用双链特异性核酸酶(DSN)消化的方法。以裂解适配法产生的Illumina HiSeq文库中的酿酒酵母rRNA序列为靶点，我们发现PDD可以有效地去除rRNA。作为文库插入大小和DSN切割要求的函数，探针产生扩展的耗尽区。使用完整的总RNA作为起始材料，探针可以在最小预期文库大小减去20个核苷酸的情况下间隔，以实现持续耗尽。当比较PDD处理的文库和未处理的文库时，没有检测到偏离靶标的偏差。我们还提供了一个生物信息学工具来设计合适的PDD探针组。我们发现，PDD是一种快速的程序，可以有效和特定地耗尽深度测序文库中的不需要的序列。由于PDD在cDNA阶段起作用，因此可以最大限度地减少对脆弱RNA样本的处理，而且进一步补救现有文库应该是可行的。重要的是，PDD保留了核苷酸拆分足迹或碱基切割研究所需的原始RNA片段边界。最后，由于PDD使用未经修饰的DNA寡核苷酸，它可以为大型项目提供一个低成本的选择，或者可以灵活地定制，以适应不同的消耗目标、样本类型和生物体。本文的在线版本(DOI：10.1186/1471-2164-15-401)包含补充材料，授权用户可以使用。
A major hurdle to transcriptome profiling by deep-sequencing technologies is that abundant transcripts, such as rRNAs, can overwhelm the libraries, severely reducing transcriptome-wide coverage. Methods for depletion of such unwanted sequences typically require treatment of RNA samples prior to library preparation, are costly and not suited to unusual species and applications. Here we describe Probe-Directed Degradation (PDD), an approach that employs hybridisation to DNA oligonucleotides at the single-stranded cDNA library stage and digestion with Duplex-Specific Nuclease (DSN). Targeting Saccharomyces cerevisiae rRNA sequences in Illumina HiSeq libraries generated by the split adapter method we show that PDD results in efficient removal of rRNA. The probes generate extended zones of depletion as a function of library insert size and the requirements for DSN cleavage. Using intact total RNA as starting material, probes can be spaced at the minimum anticipated library size minus 20 nucleotides to achieve continuous depletion. No off-target bias is detectable when comparing PDD-treated with untreated libraries. We further provide a bioinformatics tool to design suitable PDD probe sets. We find that PDD is a rapid procedure that results in effective and specific depletion of unwanted sequences from deep-sequencing libraries. Because PDD acts at the cDNA stage, handling of fragile RNA samples can be minimised and it should further be feasible to remediate existing libraries. Importantly, PDD preserves the original RNA fragment boundaries as is required for nucleotide-resolution footprinting or base-cleavage studies. Finally, as PDD utilises unmodified DNA oligonucleotides it can provide a low-cost option for large-scale projects, or be flexibly customised to suit different depletion targets, sample types and organisms. The online version of this article (doi: 10.1186/1471-2164-15-401) contains supplementary material, which is available to authorized users.
登录
查看更多内容
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
12.3
作者：
Giannoukos G;Ciulla DM;Huang K;Haas BJ;Izard J;Levin JZ;Livny J;Earl AM;Gevers D;Ward DV;Nusbaum C;Birren BW;Gnirke A
通讯作者：
Gnirke A
影响因子：
4.8
作者：
Murigneux, Valentine;Sauliere, Jerome;Le Hir, Helve
通讯作者：
Le Hir, Helve
影响因子：
4.8
作者：
Ule, J;Jensen, K;Darnell, RB
通讯作者：
Darnell, RB
影响因子：
3.7
作者：
Morlan JD;Qu K;Sinicropi DV
通讯作者：
Sinicropi DV