Thu, 12/05/2013
Customer is a cancer biologist in Harvard Medical School, and he asks about analysis of some CLIP-seq data he has.
Thu, 12/05/2013 at 5:41 PM
AccuraScience LB: Is this for Argonaute or for another RNA-binding protein? Are you interested in identifying miRNA targets or there are different purposes of your experiments? Do you have control experiments, and do you want to analyze crosslink-induced mutations?
Thu, 12/05/2013 at 6:07 PM
Customer: This is for Argonaute2 associated RNAs from nuclear and cytoplasmic fractions from three different cellular conditions of a human cancer cell line. We have done it exactly as the Darnell and Sharp groups had in their published HITS-CLIP papers-their protocols and controls. Could you please specify what control experiments you are looking for. We are interested in identifying microRNA targets. We have replicates for each and RNA-seq samples to normalize against as the total/Input amounts.
Sun, 12/08/2013 at 5:26 PM
AccuraScience LB: By control experiment, I was referring to the RNA-seq from the same sample - your description suggests that you have it.
Our routine CLIP-seq analysis procedure would include the following steps: 1) Pro-processing of reads, using FASTX-Toolkit, 2) Quality filtering, 3) Mapping of reads to the reference genome, using Novoalign (which is more proper to use than BWA because it handles indels better - indels are frequently introduced by the cross-link), 4) Peak calling using CLIPper or Pyicos, and producing output with RNA-seq as control, and 5) Motif enrichment analysis, producing list of enriched short motifs (~7-mers) in the peaks identified.
You might also be interested in extracting information about the miRNAs involved. Your CLIP-seq data may or may not have miRNA sequences - in either case, we might try to "deconvolve" the miRNAs involved starting from the 7-mer enriched motifs and the seed-match principles for miRNA targeting.
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Note: LB stands for Lead Bioinformatician. An AccuraScience LB is a senior bioinformatics expert and leader of an AccuraScience data analysis team.
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