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Question: TopHat align summary
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gravatar for Diana Afonso
2.2 years ago by
Diana Afonso • 10
Portugal/Braga/ICVS
Diana Afonso • 10 wrote:

Hello,

I am using galaxy to analyze RNA seq of 100bp sing end data, sequenced with Illumina 2500. I started by uploading bam files and using the tool "Convert from BAM to FastQ" to convert my data into FastQ format. Next I used "FastQ groomer" to convert my data into one format that contains Sanger-scaled quality values with ASCII. Finally I used TopHat with the default settings and the align summary is the following:

Reads: Input : 14520796 Mapped : 6298973 (43.4% of input) of these: 1080332 (17.2%) have multiple alignments (0 have >20) 43.4% overall read mapping rate.

I think the percentage of mapped sequences is very low. Could you please give me some tips on how I should alter the TopHat parameters to improve my results?

Thank you, Diana
fastq tophat alignment parameters rna-seq • 1.2k views
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modified 2.2 years ago by Jennifer Hillman Jackson ♦ 25k • written 2.2 years ago by Diana Afonso • 10
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gravatar for Jennifer Hillman Jackson
2.2 years ago by
Jennifer Hillman Jackson ♦ 25k
United States
Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

Double check the fastq quality score formatting. This is how: https://wiki.galaxyproject.org/Support#FASTQ_Datatype_QA

It would be a good idea to run FastQC again if you make adjustments to the reads. That report will alert you to quality problems within the data.

After that, this RNA-seq tutorial might be useful, along with the Tophat manual:

Thanks, Jen, Galaxy team
ADD COMMENT • link written 2.2 years ago by Jennifer Hillman Jackson ♦ 25k
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