Keth-seq Analysis workflow

Keth-seq是一种研究RNA二级结构的测序方法。它的文库和icSHAPE非常相似，因而我们也可以使用icSHAPE pipeline来分析Keth-seq数据，本文档示例了我们的测试结果。

流程

分析流程请参考icSHAPE-pipe

测试数据

测试数据来自GSE122096，包含如下四个样本：

      Sample Treatment Replicate
1 GSM3454818  kethoxal      rep1
2 GSM3454819  kethoxal      rep2
3 GSM3454820  no treat      rep1
4 GSM3454821  no treat      rep2

处理过程

Collapsing the reads:

readCollapse.pl  -U sample.fastq -o sample_rmdup.fq -f sample_collapse.fa

Removing adapters:

trimming.pl -U sample_rmdup.fq -o sample_trimmed.fq -l 13 -t 0 -c phred33 -a adapter.fa -m 0

Mapping:

bowtie --sam-nohead --quiet -p 5 -S --un sample_rmrrna.fq sample_trimmed.fq sample_rmrna.sam
bowtie --quiet -p 5 -S genome sample_rmrrna.fq sample.sam

Calculating RT signals:

calcRT.pl -i sample.sam -o sample.rt -r sample.rpkm -c 5

Combining RT signals:

combineRTreplicates.pl -i sample_rep1.rt:sample_rep2.rt -o sample_combine.rt

Normalized RT signals:

normalizeRTfile.pl -i sample_combine.rt -o sample_normed.rt -m mean:vigintile2 -d 32 -l 32

Calculating reactivity score:

calcEnrich.pl -f sample_case_normed.rt -b sample_control_normed.rt -o sample_enrich.out -w factor5:scaling1 -x 0.25 -y 10

Filtering reactivity score:

filterEnrich.pl -i sample_enrich.out -o sample_enrich_filter.out -t 200 -T 2 -s 5 -e 30

Visualization:

enrich2Bedgraph.pl -i sample.out -o sample.bdg -g gtf -a fasta

sort -k1,1 -k2,3n sample.bdg >sample_sorted.bdg

uniqueTrack.pl sample_sort.bdg sample_uniq.bdg

cut -f1-4 sample_uniq.bdg | grep -v NULL > sample_sim.bdg

bedGraphToBigWig sample_sim.bdg genome.chr.size sample_sim.bw

一致性检验

我们比较了论文中发表的结果（用paper代表）和本次测试结果（用kethoxal代表），两者高度吻合：

参考文献

  • [1] Li P, Shi R, Zhang Q C. icSHAPE-pipe: A comprehensive toolkit for icSHAPE data analysis and evaluation[J]. Methods, 2019.
  • [2] R.C. Spitale, R.A. Flynn, Q.C. Zhang, P. Crisalli, B. Lee, J.W. Jung, H.Y. Kuchelmeister, P.J. Batista, E.A. Torre, E.T. Kool, H.Y. Chang, Structural imprints in vivo decode RNA regulatory mechanisms, Nature 519 (7544) (2015) 486–490.
  • [3] Xu, Z. & Culver, G.M. In Methods in Enzymology; Biophysical, Chemical, and Functional Probes of Rna Structure, Interactions and Folding, Pt A (ed. Herschalag, D.) Vol 468, 47–165 (Academic Press, 2009).
  • [4] Weng X , Gong J , Chen Y , et al. Keth-seq for transcriptome-wide RNA structure mapping[J]. Nature Chemical Biology, 2020, 16(5):1-4.
  • [5] Spitale, R. C. et al. Structural imprints in vivo decode RNA regulatory mechanisms. Nature 519, 486–490 (2015).