Question: Very low concordant rate using TopHat2
gravatar for AT
5.4 years ago by
United States
AT0 wrote:

I have paired end 76 bp reads for RNA seq data from FFPE samples and I am using tophat2 (version 2.1.0) using mostly default options:

>> tophat-2.1.0.Linux_x86_64/tophat -p 8 --library-type fr-secondstrand -o . hg19 test_R1.fastq.gz test_R2.fastq.gz

and I get very low concordant rate, as given by the align_summary.txt file:

Left reads:

          Input     :  41740697

           Mapped   :  20342583 (48.7% of input)

            of these:   6651856 (32.7%) have multiple alignments (51919 have >20)

Right reads:

          Input     :  41740697

           Mapped   :  18876112 (45.2% of input)

            of these:   6285887 (33.3%) have multiple alignments (51879 have >20)

Unpaired reads:

          Input     :     51310

           Mapped   :     13565 (26.4% of input)

            of these:      2966 (21.9%) have multiple alignments (0 have >20)

47.0% overall read mapping rate.

Aligned pairs:  10618677

     of these:   4367774 (41.1%) have multiple alignments

                10544908 (99.3%) are discordant alignments

 0.2% concordant pair alignment rate.

These reads have not been trimmed at the first pass, though FASTQC did show a few reads with length as low as 0. I am running on another set of trimmed fastq file in which I have removed all reads less than a length of 20, and am waiting to see the results. Is there some other parameter that I should be setting to get better concordance..this seems very low. Any help/suggestions will be appreciated.

rna-seq tophat • 1.9k views
ADD COMMENTlink modified 5.4 years ago • written 5.4 years ago by AT0

Have a look at your command line, your second file is named test_R.fastq.gz. Shouldn't that be test_R2.fastq.gz

ADD REPLYlink written 5.4 years ago by thackl2.8k

That was a typo when putting in the question..

ADD REPLYlink written 5.4 years ago by AT0

Yeah, would have been too easy...

ADD REPLYlink written 5.4 years ago by thackl2.8k

I have been experiencing similar issues with my paired data. Did you ever figure out what the issue was for your data?

ADD REPLYlink written 5.2 years ago by olive121280
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