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  • 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,:

    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:
    fr-unstranded (default)

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

    appreciate your help.


  • #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...


    • #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 (
      [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 |...........|

      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.


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