Seqanswers Leaderboard Ad

Collapse
X
 
  • Filter
  • Time
  • Show
Clear All
new posts
  • panos_ed
    Member
    • May 2010
    • 11

    How should a strand-specific experiment look like in IGV?

    I have a paired-end, strand-specific dataset and used Trinity to assemble the contigs. I then mapped the reads back to the contigs to calculate transcript abundance and viewed the resulting bam file in IGV (don't have a reference genome).

    Is there a way that I can tell whether my dataset is really strand-specific just by looking at the picture in IGV? If I had single-end sequences I should expect to have reads only in one strand (i.e. pointing to the right). Am I right?

    But since I have paired-end reads, what the IGV picture should look like?
  • Jim Robinson
    Member
    • May 2009
    • 75

    #2
    Hi,

    It depends on the library details, but you should could try "color by first-in-pair" from the popup menu. This will color by the strand of the template, rather than the read, at least for libraries I am familiar with. Also, igvtools has options to compute coverage by pair strand, although this isn't available in igv itself yet. Better strand support is on our radar.

    Jim

    Comment

    • panos_ed
      Member
      • May 2010
      • 11

      #3
      Thanks Jim!

      I selected "first-of-pair" as you told me but can't find out what the colors mean (and couldn't find it either in IGV's manual).

      Can you please explain what the colors (red, blue and grey) mean?

      Comment

      • Jim Robinson
        Member
        • May 2009
        • 75

        #4
        red = neg strand (first read of the pair is no the neg strand)
        blue = pos strand
        grey = pair information not available, so "first of pair" is undefined

        Comment

        • panos_ed
          Member
          • May 2010
          • 11

          #5
          Thanks for the info Jim!

          So if most of the pairs in a contig contain reads that are like this:

          Code:
          =====>----------<=====
          (red)           (blue)
          then this means that sequencing was strand-specific.

          Am I right?

          What if both of them are red or blue?

          Comment

          • Simon Anders
            Senior Member
            • Feb 2010
            • 995

            #6
            No, even in a non-strand-specific library, the two reads of a pair should point towards each other (or away from each other if it was a library-prep protocol with circularization). You should check whether it is predominantly the first read that point in the sense direction of the gene it aligns to.

            Comment

            • Dario1984
              Senior Member
              • Jun 2011
              • 166

              #7
              Originally posted by Jim Robinson View Post
              red = neg strand (first read of the pair is no the neg strand)
              blue = pos strand
              grey = pair information not available, so "first of pair" is undefined
              If I have Illumina directional RNA-seq data, should the colours be the opposite or the same as the strand of the RefSeq gene, when colouring by the first read of the pair ? My colours are opposite to the RefSeq gene, but when isoforms are assembled by Cufflinks with --library-type fr-secondstrand, the isoforms are, as expected, on the same strand as the gene.

              Comment

              • stanikay
                Junior Member
                • Oct 2013
                • 4

                #8
                So is directional RNA-seq the same as strand-specific RNA-seq? any clarification will be greatly appreciated.

                Thanx

                Comment

                • panos_ed
                  Member
                  • May 2010
                  • 11

                  #9
                  According to this, directional and strand-specific are the same.

                  Comment

                  • stanikay
                    Junior Member
                    • Oct 2013
                    • 4

                    #10
                    Thanx panos-ed..

                    Comment

                    • ArghavanAri
                      Junior Member
                      • Apr 2014
                      • 2

                      #11
                      Hi,

                      I am trying to align and count the reads of paired-end strand-specific RNA-seq data using Hisat and htseq-count.
                      This is how my IGV look like. https://www.dropbox.com/s/snpapn051b...ness.jpeg?dl=0
                      (Color alignment by "first-of-pair strand") (view as pairs).
                      I am confused now. Why all the read pairs are red for NFE2L2? and clur for HNRNPA3?
                      I know that my data is strand specific, and that this color is showing the first pair read. So why both pairs have the same color?
                      Also can anyone tell me what options I should use for Hisat (or Tophat2) and htseq-count?

                      Thanks,
                      Last edited by ArghavanAri; 09-14-2015, 07:18 AM.

                      Comment

                      • cliffbeall
                        Senior Member
                        • Jan 2010
                        • 144

                        #12
                        The two genes are on different strands, you can see by the little chevrons in the annotation track at bottom, so that is expected.

                        Comment

                        • gringer
                          David Eccles (gringer)
                          • May 2011
                          • 845

                          #13
                          As others have mentioned, you need to consider the direction of mapping as well as the read number. For a standard Illumina strand-specific sample preparation, the reverse read is the first read:
                          • Reverse mapping, First read -- consistent with a gene model reading left to right on IGV
                          • Reverse mapping, Second read -- consistent with a gene model reading right to left on IGV
                          • Forward mapping, First read -- consistent with a gene model reading right to left on IGV
                          • Forward mapping, second read -- consistent with a gene model reading left to right on IGV


                          Just in case you weren't aware, Tablet (since v1.14.10.20) has a concordant / directional colouring mode that colours sequences appropriately.
                          Last edited by gringer; 09-14-2015, 07:31 PM.

                          Comment

                          • ArghavanAri
                            Junior Member
                            • Apr 2014
                            • 2

                            #14
                            Thanks for the information.
                            But I still cannot understand the difference between fr-firststrand & fr-secondstrand in Tophat2 (or RF & FR in Hisat).
                            Can anyone help me with that?

                            Comment

                            • cliffbeall
                              Senior Member
                              • Jan 2010
                              • 144

                              #15
                              Originally posted by ArghavanAri View Post
                              Thanks for the information.
                              But I still cannot understand the difference between fr-firststrand & fr-secondstrand in Tophat2 (or RF & FR in Hisat).
                              Can anyone help me with that?
                              I am fairly sure fr-firststrand means read one (aka forward read or fr) corresponds to the reference genome sequence (firststrand). fr-secondstrand means read one corresponds to the reverse complement (secondstrand) of the reference genome. FR and RF would be a similar concept - but they are just making a shorthand where if the read one or forward read corresponds to the reference strand it maps to the left of the reverse read on a linear representation of the genome (FR) and if it corresponds to the reverse complement of the reference it maps to the right of the reverse (RF).

                              I hope that makes sense -- you might need to go back to DNA/genome structure and how Illumina works to really understand the above.

                              Comment

                              Latest Articles

                              Collapse

                              • seqadmin
                                Pathogen Surveillance with Advanced Genomic Tools
                                by seqadmin




                                The COVID-19 pandemic highlighted the need for proactive pathogen surveillance systems. As ongoing threats like avian influenza and newly emerging infections continue to pose risks, researchers are working to improve how quickly and accurately pathogens can be identified and tracked. In a recent SEQanswers webinar, two experts discussed how next-generation sequencing (NGS) and machine learning are shaping efforts to monitor viral variation and trace the origins of infectious...
                                03-24-2025, 11:48 AM
                              • seqadmin
                                New Genomics Tools and Methods Shared at AGBT 2025
                                by seqadmin


                                This year’s Advances in Genome Biology and Technology (AGBT) General Meeting commemorated the 25th anniversary of the event at its original venue on Marco Island, Florida. While this year’s event didn’t include high-profile musical performances, the industry announcements and cutting-edge research still drew the attention of leading scientists.

                                The Headliner
                                The biggest announcement was Roche stepping back into the sequencing platform market. In the years since...
                                03-03-2025, 01:39 PM

                              ad_right_rmr

                              Collapse

                              News

                              Collapse

                              Topics Statistics Last Post
                              Started by seqadmin, 03-20-2025, 05:03 AM
                              0 responses
                              49 views
                              0 reactions
                              Last Post seqadmin  
                              Started by seqadmin, 03-19-2025, 07:27 AM
                              0 responses
                              57 views
                              0 reactions
                              Last Post seqadmin  
                              Started by seqadmin, 03-18-2025, 12:50 PM
                              0 responses
                              50 views
                              0 reactions
                              Last Post seqadmin  
                              Started by seqadmin, 03-03-2025, 01:15 PM
                              0 responses
                              201 views
                              0 reactions
                              Last Post seqadmin  
                              Working...