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Question: Rnastar judging successful result
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gravatar for mbt
11 weeks ago by
mbt • 0
mbt • 0 wrote:

Howdy RNA seq files are quite large ca. 2 gbytes fastq compressed. Paired end. I’ve used rnastar. Tried to map to hg38 used whole used whole human genome annotation gtf file from ucsf genome browser. Average read size 124 bp, average fragment size 450 bp. For rnastar junction overlap used 111 bp.

These are results from log file. Looks like a lot of the reads were unmapped? I got a similar result from top hat. 

2   3   4
N_unmapped  23359811    23359811    23359811
N_multimapping  386274  386274  386274
N_noFeature 193779  2056977 202263
N_ambiguous 1867858 44759   1853764
uc031tla.1  0   0   0

I’m having trouble judging the quality of the alighment. I used rnastar because I heard it is supposed to be faster and just as sensitive as hisat. Qc of reads looks good. Maybe the problem is with the reference genome/gtf file.

Greatly appreciate your answers to previous questions. Mike (mbt5766)
rna-seq • 79 views
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modified 11 weeks ago by Jennifer Hillman Jackson ♦ 25k • written 11 weeks ago by mbt • 0
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gravatar for Jennifer Hillman Jackson
11 weeks ago by
Jennifer Hillman Jackson ♦ 25k
United States
Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

Related post: https://biostar.usegalaxy.org/p/29348/

What happens when you map without a reference GTF?

Did you also map with a reference GTF with Tophat (and restrict the output to known splice-junctions)?

Are you sure the NGS reads are human? Are they in fastqsanger or fastqsanger.gz format? Run FastQC to check.

If all true, the reference annotation may be a mismatch for the hg38 genome build. Maybe try one that is known to be a match?

There are at least two good choices to test with: Genecode Genes and iGenomes. See this prior post for details about how to get these into Galaxy: https://biostar.usegalaxy.org/p/29343/#29347

FAQs: https://galaxyproject.org/support/#getting-inputs-right

  • Mismatched Chromosome identifiers (and how to avoid them)
  • How to format fastq data for tools that require .fastqsanger format?

Thanks! Jen, Galaxy team
ADD COMMENT • link written 11 weeks ago by Jennifer Hillman Jackson ♦ 25k
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