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  • Cuffdiff error [bam_header_read] invalid BAM binary header (this is not a BAM file).

    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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