Tophat mapping problem on reads with length=50nt(or 51nt)

[tinkering]

Hi,

I used tophat version 2.0.10 to map a fastq file with reads (=51nt) to human reference genome with the following command:
tophat -p 4 --library-type fr-firststrand --GTF genes.gtf -o output_tophat --no-novel-juncs ./genome_references/bowtie2_hg19/hg19 h11648.fq[fastq]

It failed after running through building the bowtie index, the log is as follows:
[2015-10-14 13:36:10] Beginning TopHat run (v2.0.10)
-----------------------------------------------
[2015-10-14 13:36:10] Checking for Bowtie
Bowtie version: 2.1.0.0
[2015-10-14 13:36:10] Checking for Samtools
Samtools version: 0.1.18.0
[2015-10-14 13:36:10] Checking for Bowtie index files (genome)..
[2015-10-14 13:36:10] Checking for reference FASTA file
[2015-10-14 13:36:10] Generating SAM header for /data/Mullen_1/Data/genome_references/bowtie2_hg19/hg19
[2015-10-14 13:36:23] Reading known junctions from GTF file
[2015-10-14 13:36:27] Preparing reads
left reads: min. length=51, max. length=51, 2912 kept reads (0 discarded)
[2015-10-14 13:36:27] Building transcriptome data files h11648_tophat/tmp/H148hrlncRNA_UCSCgenes
[2015-10-14 13:36:47] Building Bowtie index from H148hrlncRNA_UCSCgenes.fa
[2015-10-14 13:48:08] Mapping left_kept_reads to transcriptome genes with Bowtie2
/apps/source/tophat/2.0.10/bam2fastx: /lib64/libz.so.1: no version information available (required by /apps/source/tophat/2.0.10/bam2fastx)
/apps/source/tophat/2.0.10/fix_map_ordering: /lib64/libz.so.1: no version information available (required by /apps/source/tophat/2.0.10/fix_map_ordering)
[FAILED]
Error running:
/apps/source/tophat/2.0.10/bam2fastx --all h11648_tophat/tmp/left_kept_reads.bam|/apps/source/bowtie2-2.1.0/bowtie2-2.1.0/bowtie2-align -k 60 -D 15 -R 2 -N 0 -L 20 -i S,1,1.25 --gbar 4 --mp 6,2 --np 1 --rdg 5,3 --rfg 5,3 --score-min C,-14,0 -p 4 --sam-no-hd -x h11648_tophat/tmp/genes -|/apps/source/tophat/2.0.10/fix_map_ordering --bowtie2-min-score 15 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --sam-header h11648_tophat/tmp/H148hrlncRNA_UCSCgenes.bwt.samheader.sam - - h11648_tophat/tmp/left_kept_reads.m2g_um.bam | /apps/source/tophat/2.0.10/map2gtf --sam-header h11648_tophat/tmp/hg19_genome.bwt.samheader.sam h11648_tophat/tmp/genes.fa.tlst - h11648_tophat/tmp/left_kept_reads.m2g.bam > h11648_tophat/logs/m2g_left_kept_reads.out


I used the same parameters of tophat to run through single-end reads with 42 nt length, but cannot know what made the tophat run through single-end reads with 51nt. Even after trimming the reads into 50nt, it did not work. And it also failed on small dataset.

Does anyone have any suggestions on solving this problem?

Many thanks!

[GenoMax]

Two things. You are using an older version of tophat. Where possible you should always use the latest.

You also appear to have a library version mismatch (/lib64/libz.so.1). What OS are you using?

[tinkering]

Hi GenoMax,

Thanks for your reply!
I ran the Tophat on the OS: Red Hat Enterprise Linux Server release 6.5 (Santiago). Regarding to the library, if this is a problem, the Tophat ran through on other data smoothly.

Thanks again!

[GenoMax]

@tinkering: I have amended my post #2. It appears that the version of lib64/libz available on your system is dissimilar from the one tophat2 is expecting (I think you are using the pre-compiled binary? You can check by running "ldd -v tophat2" to see what libraries are linked). If you were to compile from source that message should go away since the dynamic linker will use the library available on your system.

There are multiple commands separated by pipes in the error you include above. Have you tried to run them independently to see where the error is coming from? What is the exact error BTW?