Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Align paired and unpaired reads with Tophat

    I have a stranded PE RNAseq data set that I want to align with tophat using the --library-type fr-firststrand option. After adaptor trimming I end up with my 4 files:

    paired_1
    paired_2
    unpaired_1
    unpaired_2

    Is there a way to align these so I do not loose the strand information for the unpaired reads? When I run tophat with the files listed like this:

    paired_1,unpaired_1 paired_2,unpaired_2

    It seems to want to try and align the two unpaired files as paired files. If I combine the two unpaired files and run tophat with the files listed like this:

    paired_1 paired_2,unpaired

    It recognizes the last file as unpaired. But am I loosing my strand specificity by aligning this way? Thanks.

  • #2
    Inputting the files like:
    Code:
    paired_1,unpaired_1 paired_2,unpaired_2
    Will result in "unpaired_1" and "unpaired_2" being treated as paired, which is the opposite of what you want. To keep the strandedness correct you'll need to run things twice. Firstly using "paired_1,unpaired_1 paired_2" with library-type set to fr-firststrand and then just "unpaired_2" by itself with "fr-secondstrand". I should note that aligning unpaired_2 is usually not worthwhile (the reads are often crap), but perhaps you'll get luckier than I have with that.

    Comment


    • #3
      Thank you very much for your response, that makes a lot of sense.

      Comment


      • #4
        On this note ..I Have a question. I am doing PE RNA-Seq analysis of mouse data. My read length is 90bp and fragment size is 127 bp which means I have overlapping reads. I used Flash and found that not all reads overlap.

        I have a file with merged reads which overlaps and also two files with non overlap reads. basically three fastq files.

        How do I go about running Tophat with this and I am not really sure hot to calculate the mean inner mate distance and sd??

        Has anyone come across this situation???

        Comment


        • #5
          There's no need to run Flash on them, just use tophat2 and bowtie2 instead of bowtie1.

          For the mean inner distance, just try 0 and see if that produces acceptable results (I recall reading that tophat re-estimates the insert length as it runs, though I can't say I've ever checked if that's correct).

          Comment


          • #6
            Thanks alot Ryan... I will try that.

            Comment


            • #7
              or just use STAR that do not need to specify inner distance. and also is much faster for the same ( even better ) results

              Comment

              Latest Articles

              Collapse

              • seqadmin
                Best Practices for Single-Cell Sequencing Analysis
                by seqadmin



                While isolating and preparing single cells for sequencing was historically the bottleneck, recent technological advancements have shifted the challenge to data analysis. This highlights the rapidly evolving nature of single-cell sequencing. The inherent complexity of single-cell analysis has intensified with the surge in data volume and the incorporation of diverse and more complex datasets. This article explores the challenges in analysis, examines common pitfalls, offers...
                06-06-2024, 07:15 AM
              • 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

              ad_right_rmr

              Collapse

              News

              Collapse

              Topics Statistics Last Post
              Started by seqadmin, Today, 07:24 AM
              0 responses
              9 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, Yesterday, 08:58 AM
              0 responses
              11 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, 06-12-2024, 02:20 PM
              0 responses
              16 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, 06-07-2024, 06:58 AM
              0 responses
              182 views
              0 likes
              Last Post seqadmin  
              Working...
              X