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2021-02-23根据RNA-seq测序数据判断文库类型和链特异性

2021-02-23根据RNA-seq测序数据判断⽂库类型和链特异性

药盒印刷⽬录

问题描述

如何只根据fastq⽂件确定RNA-seq的建库⽅法和链特异性？

关键词

strandedness，strandness，strand-specific，library type，library construction，RNA-seq

解决⽅案

⽐对fastq数据，⽣成sam或bam⽂件

Mapping your RNA-seq reads as if they were non-strand specific

准备bed格式的基因组注释⽂件

可下载gtf⽂件，然后使⽤bedops的gtf2bed将其转化为bed格式

conda install bedops

吊耳gtf2bed < f > ABC.bed

运⾏RSeQC的infer-experiment.py

conda install rseqc

infer_experiment.py -r ABC.bed -i sample.bam

输出结果为4⾏，例如：

This is PairEnd Data

Fraction of reads failed to determine: 0.0072

Fraction of reads explained by "1++,1--,2+-,2-+": 0.9441

Fraction of reads explained by "1+-,1-+,2++,2--": 0.0487

根据结果确定⽂库类型

对于双端测序，1++,1--,2+-,2-+与1+-,1-+,2++,2--的⽐例可以体现出⽂库类型；对于单端测序，++,--和+-,-+的⽐例亦然：⼆者⽐例接近1时，⽂库为unstranded，不具有链特异性，建库⽅法包括Standard Illumina, NuGEN OvationV2等

前者明显⼤于后者时，⽂库为FR/fr-secondstrand stranded，建库⽅法包括dUTP, NSR, NNSR, Illumina TruSeq Strand Specific Total RNA, NEBNext Ultra II Directional等

后者明显⼤于前者时，⽂库为RF/fr-firststrand stranded，建库⽅法包括 Ligation, Standard SOLiD, NuGEN Encore等

⽂库类型对应的软件参数

Tool RF/fr-firststrand stranded FR/fr-secondstrand stranded Unstranded check_strandedness (output)RF/fr-firststrand FR/fr-secondstrand unstranded

IGV (5p to 3p read orientation

code)F2R1F1R2

F2R1 or

F1R2

TopHat (–library-type

parameter)fr-firststrand fr-secondstrand

fr-

unstranded

过氧化氢浓度测定

HISAT2 (–rna-strandness

parameter)

R/RF F/FR NONE

HTSeq (–stranded/-s

parameter)

reverse yes no

Picard CollectRnaSeqMetrics

(STRAND_SPECIFICITY

parameter)

隐藏滑轨

SECOND_READ_TRANSCRIPTION_STRAND FIRST_READ_TRANSCRIPTION_STRAND NONE Kallisto quant (parameter)–rf-stranded–fr-stranded NONE StringTie (parameter)–rf–fr NONE FeatureCounts (-s parameter)210

directdraw

RSEM (–forward-prob

parameter)

010.5

Salmon (–libType parameter)ISR (assuming paired-end with inward read

orientation)

ISF (assuming paired-end with inward

read orientation)

IU (assuming

paired-end

with inward

read

orientation)

typec转usbTrinity (–SS_lib_type parameter)RF FR NONE

MGI CWL YAML (strand

parameter)first second NONE

Tool RF/fr-firststrand stranded FR/fr-secondstrand stranded Unstranded

⼀个傻⽠式软件

参考

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