Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • RSeqQC infer_experiment strandedness

    Hi,

    I am using RSeqQC infer_experiment.py to look at whether or not a library is stranded and I got the following results:

    This is SingleEnd Data
    Fraction of reads failed to determine: 0.0242
    Fraction of reads explained by "++,--": 0.0398
    Fraction of reads explained by "+-,-+": 0.9360


    What does this mean? it appears that the reads are mapping to the reverse complement strand relative to the gene, what kind of sequencing protocol would do this? Does it have a name?

    Thanks for any help,
    Leanne

  • #2
    Hi Leanne,

    The result shows you, that you have a stranded library. It also means, as you already said, that the reads are the reverse complement of the transcripts.
    A couple of library preps produce reads in that flavour:
    TruSeq stranded, ScriptSeq, Sense, ....
    Just use the strandedness as detected in your analyses (e.g. htseq-count ... -s reverse ..).
    Cheers,
    Michael

    Comment


    • #3
      The "+-,-+" syntax is described here and has got to be about the most cryptic way of conveying things that's possible. What this means is that you have strand-specific data made with the dUTP method. This is the most common type these days and means that if you need to do counting, that you should use the "-s reverse" option in htseq-count or "-s 2" in featureCounts. If you happen to be using tophat2 for alignment, you want the "fr-firststrand" library type.

      Edit: I should have refreshed, Michael beat me to it!

      Comment


      • #4
        Thanks guys! this was very helpful! The paper says an Illumina TruSeq Stranded mRNA LT kit so based on what Michael said it is to be expected that the read are the reverse complement of the gene. Another sequencing set from the same paper ( which was also indicated to use the Illumina TruSeq Stranded mRNA kit) gave me the following RSeqQC results:


        Fraction of reads explained by "++,--": 0.4775
        Fraction of reads explained by "+-,-+": 0.4956

        Does this mean that maybe the strandedness part of the kit didn't work as well? Or maybe a non stranded kit was used?

        Comment


        • #5
          Either the paper was wrong or their kit failed completely. My guess is that the paper was wrong and that they used an unstranded kit.

          Comment


          • #6
            Thanks dpryan!

            I do have another question if multiple alignments is specified in the bowtie alignment (-a) will infer_experiment be able to work correctly in predicting strandedness?

            Comment


            • #7
              Most likely it'll still work, though the percentages will be different.

              Comment

              Latest Articles

              Collapse

              • 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
              • seqadmin
                Essential Discoveries and Tools in Epitranscriptomics
                by seqadmin




                The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist...
                04-22-2024, 07:01 AM

              ad_right_rmr

              Collapse

              News

              Collapse

              Topics Statistics Last Post
              Started by seqadmin, 05-14-2024, 07:03 AM
              0 responses
              19 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, 05-10-2024, 06:35 AM
              0 responses
              44 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, 05-09-2024, 02:46 PM
              0 responses
              54 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, 05-07-2024, 06:57 AM
              0 responses
              42 views
              0 likes
              Last Post seqadmin  
              Working...
              X