2021-02-23根据RNA-q测序数据判断⽂库类型和链特异性
⽬录
问题描述
如何只根据fastq⽂件确定RNA-q的建库⽅法和链特异性？
关键词
strandedness，strandness，strand-specific，library type，library construction，RNA-q
解决⽅案
⽐对fastq数据，⽣成sam或bam⽂件
Mapping your RNA-q 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
gtf2bed < f > ABC.bed
运⾏RSeQC的infer-experiment.py
conda install rqc
状语后置句infer_experiment.py -r ABC.bed -i sample.bam
脉脉含情的读音输出结果为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-condstrand 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-condstrand stranded Unstranded check_strandedness (output)RF/fr-firststrand FR/fr-condstrand unstranded
IGV (5p to 3p read orientation
code)F2R1F1R2钱钟书经典语录
普吉岛自由行攻略
F2R1 or
F1R2
TopHat (–library-type
parameter)fr-firststrand fr-condstrand
fr-
unstranded
HISAT2 (–rna-strandness
HISAT2 (–rna-strandness
parameter)
R/RF F/FR NONE
HTSeq (–stranded/-s
parameter)
rever yes no
Picard CollectRnaSeqMetrics
(STRAND_SPECIFICITY
parameter)
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
RSEM (–forward-prob
parameter)
010.5
Salmon (–libType parameter)ISR (assuming paired-end with inward read
多吃蔬菜orientation)
ISF (assuming paired-end with inward
read orientation)
IU (assuming
paired-end
身不由己什么意思
with inward
read
orientation)
阳光明媚的近义词Trinity (–SS_lib_type parameter)RF FR NONE
MGI CWL YAML (strand
parameter)first cond NONE
Tool RF/fr-firststrand stranded FR/fr-condstrand stranded Unstranded
蕃茄鸡蛋汤
⼀个傻⽠式软件
参考
