Question: GEO SRA fastq-dump with very low mapping rate (Galaxy)
gravatar for human
6 days ago by
human0 wrote:

Dear Biostars,

I am a quite unexperienced biologist doing a metaanalysis of RNA-seq/microarray expression data using Galaxy and I get very low mapping percentage (0.5-1.7 %) with one particular GEO dataset. I have tested 3 other Datasets and mapping with BWA (same parameters) works like a charm (80-90 % mapping).

I import the GEO datasets into Galaxy using NCBI SRA Tools, when you look at the reads quality formats they look different than other GEO uploads. I am aware that there are 3 types of fastq format and this is important for downstream application.

P.S. data is not trimmed (has Illumina adapter 1 overepresented). I assumed its Illumina 1.9 encoded (FASTQC says so) AND also tried FastqGroomer covert assumed solexa format (+64) to sanger format (+33), but still low mapping. I also tried BWA, BWA Illumina, BWA-MEM, Bowtie2.

low % mapping sample is single end 35 bp sequenced on Illumina GAIIx, no idea wether demultiplexed or prepocessed. high % mapping sample is single end 35 bp sequenced on Hiseq 2000.

Is the file format wrong or do the fastq files need more pre-processing before mapping ?

If you need more info stats SRR number ect. please say so.

Low % mapping fastq file (2 reads)









Other GEO dataset good % mapping:

@D256N5M1:21:C00B5ABXX:5:1101:1108:2087 1:N:0:TGACCA




@D256N5M1:21:C00B5ABXX:5:1101:1059:2089 1:N:0:TGACCA






rna-seq alignment galaxy • 33 views
ADD COMMENTlink modified 5 days ago by Jennifer Hillman Jackson24k • written 6 days ago by human0
gravatar for Jennifer Hillman Jackson
5 days ago by
United States
Jennifer Hillman Jackson24k wrote:


Data extracted from this source, with this tool, has been re-scaled to Sanger Phred+33 format even if originally submitted in another format. This is the .fastqsanger datatype in Galaxy.

For RNA-seq reads, a splice-aware mapping tool would be a better choice. Try HISAT2.

Thanks! Jen, Galaxy team

ADD COMMENTlink written 5 days ago by Jennifer Hillman Jackson24k
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