Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • strand specific mayhem for Tophat and Cufflinks

    Hi I have a strand specific RNA-Seq experiment. The libraries were prepared based on dUTP and I was told to use forward strand.

    I used Tophat to align the fastq files and merged replicates. I get a single bam file and used RseqQC on bam file. I get the following output:

    This is PairEnd Data
    Fraction of reads failed to determine: 0.0040
    Fraction of reads explained by "1++,1--,2+-,2-+": 0.0087
    Fraction of reads explained by "1+-,1-+,2++,2--": 0.9873


    From RSeqQC documentation,:

    1+-,1-+,2++,2–
    read1 mapped to ‘+’ strand indicates parental gene on ‘-‘ strand
    read1 mapped to ‘-‘ strand indicates parental gene on ‘+’ strand
    read2 mapped to ‘+’ strand indicates parental gene on ‘+’ strand
    read2 mapped to ‘-‘ strand indicates parental gene on ‘-‘ strand


    I understood that the bam files, majority of reads, where read2 mapped to + strand with gene on + strand.

    My question:

    How do I set the option --library-type for cufflinks. Cufflinks offfers multiple options for library-type such as:

    Supported library types:
    ff-firststrand
    ff-secondstrand
    ff-unstranded
    fr-firststrand
    fr-secondstrand
    fr-unstranded (default)
    transfrags

    in my case, based on rseq qc, shoudl i choose ff-secondstrand? or fr-firststrand or fr-reversestrand ?

    appreciate your help.

    Thanks
    Adrian.

  • #2
    You should use fr-firststrand, based on the Tophat manual.

    Also see:
    Informatics for RNA-seq: A web resource for analysis on the cloud. Educational tutorials and working pipelines for RNA-seq analysis including an introduction to: cloud computing, critical file form...

    Comment


    • #3
      Dear Group,

      I never came across errors in Tophat2/Cufflinks analysis and this error is troubling me and I cannot find a proper solution. I am posting in anticipation that I could get some help.

      I have a fastq files from RNA-Seq experiment (1x50bp;strand specific dUTP) single-end 50bp reads.

      I aligned the files using tophat2.

      tophat -p 16 --library-type fr-firststrand -G /HumanGenome/Grch37/Homo_sapiens/Ensembl/GRCh37/Annotation/Archives/archive-2015-07-17-14-31-42/Genes/genes.gtf -o myDir /HumanGenome/Grch37/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome fastq1.fastq

      I get accepted_hits.bam file along with other files and I do not see any error.


      2. Next I ran, cufflinks and I did not get any problems here. I could successfully generate transcripts.gtf file.

      cufflinks -p 16 --library-type fr-firststrand -o myDirCL accepted_hits.bam

      3. I next did cuff merge using all gtf files in a list - gtfList

      cuffmerge -p 16 -g /HumanGenome/Grch37/Homo_sapiens/Ensembl/GRCh37/Annotation/Archives/archive-2015-07-17-14-31-42/Genes/genes.gtf -s /HumanGenome/Grch37/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome.fa gtfList

      4. Cuffdiff fails:
      cuffdiff -p 16 --library-type fr-firststrand -o mycfDiff /HumanGenome/Grch37/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome.fa -u merged.gtf samp1a.bam,samp1b.bam samp2a.bam,samp2b.bam


      Warning: Could not connect to update server to verify current version. Please check at the Cufflinks website (http://cufflinks.cbcb.umd.edu).
      [bam_header_read] EOF marker is absent. The input is probably truncated.
      [bam_header_read] invalid BAM binary header (this is not a BAM file).
      [16:06:53] Loading reference annotation.


      right after saying loading reference annotation, pipeline fails.


      I compared this with previous analyses. I never had EOF marker absent with successful cuffdiff runs.

      I validated using ValidateSamFile of Picard and I do not see any difference between bam files of successful cuffdiff analysis and failed analysis.

      Also Bam file is gzip file and I do see the end of bam file of failed run :

      tail accepted_hits.bam | hexdump -C
      00000980 54 84 0f 1b e3 be fa f6 50 67 5d b5 92 9c 16 24 |T.......Pg]....$|
      00000990 b6 9e 54 68 40 ff 07 3c e5 ef 1d 2f 8e 00 00 1f |..Th@..<.../....|
      000009a0 8b 08 04 00 00 00 00 00 ff 06 00 42 43 02 00 1b |...........BC...|
      000009b0 00 03 00 00 00 00 00 00 00 00 00 |...........|
      000009bb



      Can any one suggest what could be wrong here.

      I suspect, if I am correct in executing tophat for single-end 50bp, strand-specific reads.
      There are 4 fastq files for each sample.

      Header of fastq file:

      file 1 : @SN930:564:H3Y5YBCXY:1:1101:1228:2226 1:N:0:AGTCAA
      file 2: @SN930:565:H3Y5YBCXY:1:1101:1263:2151 1:N:0:AGTCAAC
      file 3: @SN930:564:H3Y5YBCXY:2:1101:1433:2070 1:N:0:AGTCAA
      file 4: @SN930:565:H3Y5YBCXY:2:1101:1282:2078 1:N:0:AGTCAAC


      thanks a lot.
      Adrian

      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, Yesterday, 06:54 AM
      0 responses
      10 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 06-14-2024, 07:24 AM
      0 responses
      16 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 06-13-2024, 08:58 AM
      0 responses
      16 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 06-12-2024, 02:20 PM
      0 responses
      17 views
      0 likes
      Last Post seqadmin  
      Working...
      X