Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • mapping strand-specific RNA-seq reads using TopHat

    I am using ScriptSeq Complete Gold Kit to generate pair-end RNAseq data, and wished to map the reads in a strand-specific manner. I referred TopHat FAQ, which suggested to try 'library-type' - 'fr-firststrand' and 'fr-secondstrand' separately, and the one resulting in many more splicing junctions in junctions.bed should be the right library type.

    I checked the forum, and people said that the kit should go with 'fr-secondstrand', but I still wished to double-check it. So I ran 'unstranded','firststrand' and 'secondstrand' separately on my read pairs, but found the results are quite unexpectedly similar. Specifically the junctions.bed and acceptedhits.bam file are pretty much of the same size for all of them. As I am using pair end, presumably if I force strand-specific mapping, at least one option above should give really poor alignment. But the alignment was quite similar, as you can see from the attached figure (from 1st to 3rd panels are first-strand, second-strand and non-stranded, respectively).

    Can you help see what's wrong here? Thanks!

    Code:
    tophat --library-type fr-secondstrand -o $3 -r $4 -p 4 -g 1 -G $GTF $Ref $read_A $read_B
    Attached Files

  • #2
    Hi,
    Unstranded library of RNASeq can have strand bias, If yes then how can it be ruled out?
    do we really look into it in case of fusion identification using tophat fusion?
    Regards
    Bharati

    Comment


    • #3
      @caswater:
      Tophat by default aligns to the genome and retains discordant mappings as well. So that would explain why your three alignments are fairly similar. A better way to check the strand specificity and library-type is using RSeqC.

      see:
      Bridged amplification & clustering followed by sequencing by synthesis. (Genome Analyzer / HiSeq / MiSeq)

      Techniques and protocol discussions on sample preparation, library generation, methods and ideas

      Comment

      Latest Articles

      Collapse

      • seqadmin
        Latest Developments in Precision Medicine
        by seqadmin



        Technological advances have led to drastic improvements in the field of precision medicine, enabling more personalized approaches to treatment. This article explores four leading groups that are overcoming many of the challenges of genomic profiling and precision medicine through their innovative platforms and technologies.

        Somatic Genomics
        “We have such a tremendous amount of genetic diversity that exists within each of us, and not just between us as individuals,”...
        05-24-2024, 01:16 PM
      • seqadmin
        Recent Advances in Sequencing Analysis Tools
        by seqadmin


        The sequencing world is rapidly changing due to declining costs, enhanced accuracies, and the advent of newer, cutting-edge instruments. Equally important to these developments are improvements in sequencing analysis, a process that converts vast amounts of raw data into a comprehensible and meaningful form. This complex task requires expertise and the right analysis tools. In this article, we highlight the progress and innovation in sequencing analysis by reviewing several of the...
        05-06-2024, 07:48 AM

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by seqadmin, 05-24-2024, 07:15 AM
      0 responses
      16 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 05-23-2024, 10:28 AM
      0 responses
      18 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 05-23-2024, 07:35 AM
      0 responses
      22 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 05-22-2024, 02:06 PM
      0 responses
      11 views
      0 likes
      Last Post seqadmin  
      Working...
      X