Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • How to get read-counts for sam files with transcriptome reference??

    Hi,

    I am working on rna-seq data analysis..
    Mapped the reads to transcriptome reference (cDNA fasta file) for wheat using Bowtie2.
    In the next step, I want to get the read counts.. I tried using featureCounts from Rsubread, but I get errors of the likes:

    "
    Warning: failed to find the gene identifier attribute in the 9th column of the provided GTF file.
    The specified gene identifier attribute is 'transcript_id'
    The attributes included in your GTF annotation are 'Parent=transcript:BAE47658;Name=BAE47658-1;constitutive=1;ensembl_end_phase=0;ensembl_phase=0;exon_id=BAE47658-1;rank=1;version=1'
    "

    But, the gff3 file i provided has trancript_id identifiers also.

    In one case, I used GTF.attrType="protein_id" & GTF.featureType="CDS".. There was no warning in this case.. But the asigned reads were 0.

    Also, featureCounts expects chromosome numbers in column 3 of sam file, but I have transcript ids as the reference was transcriptome..
    What to use to get read-counts in this case??

    Also, I wrote a perl script to count the number of times reads mapped to each of the transcripts (~15,000).. Can I use the counts obtained from this script?? Because I dont need to gather metafeatures into features and then count..

    I am really confised, please help me out!

  • #2
    Also, I wrote a perl script to count the number of times reads mapped to each of the transcripts (~15,000).. Can I use the counts obtained from this script?? Because I dont need to gather metafeatures into features and then count..
    That will work. How did you handle multi-mappers during alignment? That can skew some of your counts.

    Comment


    • #3
      I used the --very-sensitive option in Bowtie2 while alignment. The use of this option was justified in (https://www.ncbi.nlm.nih.gov/pubmed/27735125 supplementary) to get read counts for homoeologs in wheat. So, this option leads to mapping a read against the best (single) match out of highly identical sequences also..
      Is it alright or am I missing something?

      Also, generally, when taking transcripts as reference, can't featurecounts be used to get read counts? What other softwares may be used? I'll continue using the in-house perl script for now.

      Comment

      Latest Articles

      Collapse

      • seqadmin
        Strategies for Sequencing Challenging Samples
        by seqadmin


        Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
        03-22-2024, 06:39 AM
      • seqadmin
        Techniques and Challenges in Conservation Genomics
        by seqadmin



        The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

        Avian Conservation
        Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
        03-08-2024, 10:41 AM

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by seqadmin, Yesterday, 06:37 PM
      0 responses
      10 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, Yesterday, 06:07 PM
      0 responses
      9 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-22-2024, 10:03 AM
      0 responses
      49 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-21-2024, 07:32 AM
      0 responses
      67 views
      0 likes
      Last Post seqadmin  
      Working...
      X