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Old 12-03-2018, 02:57 PM   #1
VIOG
Junior Member
 
Location: London

Join Date: Dec 2018
Posts: 1
Default Very low TopHat aligment using public available dataset

Probably I'm missing something very simple, but I'm quiet new on this and maybe you can help me to sort this out.

I've downloaded a raw data from GEO (GSE70452, llumina Genome Analyzer (Mus musculus) -accordingly with the webpage information-) to analyse it but using a different pipeline than the authors did. After running FASTQC the data were trimmed using ea-utils. (See the images from before/after trimmed: , and then for the alignment I've decided tu use TopHat2 running the next command

Code:
nohup tophat -G ~/Genomes/GRCm38primary/GRCm38.gtf -p7 -o .  ~/Genomes/GRCm38primary/GRCm38 SRR2084761Q10.fastq
Unfortunately as an output I'm getting a very low alignment, marking the summary the next issues:

Reads:
Input : 95433050
Mapped : 1762944 ( 1.8% of input)
of these: 150943 ( 8.6%) have multiple alignments (3496 have >20)
1.8% overall read mapping rate.



Also, this information came out during the alignment:

[2018-11-28 16:44:56] A summary of the alignment counts can be found in ./align_summary.txt
[2018-11-28 16:44:57] Run complete: 1 days 22:31:16 elapsed
samtools view: writing to standard output failed: Broken pipe
samtools view: samtools view: writing to standard output failedwriting to standard output failed: Broken pipe
: Broken pipe
samtools view: samtools view: writing to standard output failedsamtools view: writing to standard output failed: Broken pipe
writing to standard output failed: Broken pipe
: Broken pipe
samtools view: writing to standard output failed: Broken pipe
samtools view: writing to standard output failed: Broken pipe
samtools view: writing to standard output failed: Broken pipe
samtools view: writing to standard output failed: Broken pipe
samtools view: writing to standard output failed: Broken pipe
samtools view: error closing standard output: -1
samtools view: error closing standard output: -1
samtools view: writing to standard output failed: Broken pipe
samtools view: error closing standard output: -1
samtools view: writing to standard output failed: Broken pipe
samtools view: writing to standard output failed: Broken pipe
samtools view: error closing standard output: -1
samtools view: error closing standard output: -1
samtools view: error closing standard output: -1
samtools view: error closing standard output: -1
samtools view: error closing standard output: -1
samtools view: error closing standard output: -1
samtools view: error closing standard output: -1
samtools view: error closing standard output: -1



Any thoughts? I've read that many people suggest possible samples contamination when this happen, but don't think so because at the end this data was analysed and published.

Thanks in advance
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Old 12-03-2018, 03:56 PM   #2
GenoMax
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Location: East Coast USA

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Posts: 6,813
Default

At this point there is no reason to use TopHat for RNAseq analysis. There are current options like STAR, BBMap, HISAT2 which should be used.
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