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Old 01-05-2016, 05:41 AM   #1
Location: Germany

Join Date: Jan 2013
Posts: 17
Default Problem somewhere during paired-end seq analysis -> no reads mapped

Hello everybody,

this is my fist time to try to handle to analyze paired-end sequencing files.
Finally BamCoverage complained there are no mapped reads in my Bam file. Also IGV doesn't show any reads.
I really don't know, where i lost my reads.
Here is what i did:

# Quality an adaptor trimming with trim galore:

trim_galore ../paired_1.fastq ../paired_2.fastq -q 20 --paired --phred33

# Mapping with bowtie

bowtie_opts="-p 4 -m 3 -S"
bowtie $bowtie_opts $bowtie_index -I 0 -X 700 -1 ../paired_1 .fq -2 ../paired_2.fq > paired.SAM

# Mapping report:
# reads processed: 4743277
# reads with at least one reported alignment: 1979307 (41.73%)
# reads that failed to align: 2758162 (58.15%)
# reads with alignments suppressed due to -m: 5808 (0.12%)
Reported 1979307 paired-end alignments to 1 output stream(s)

(-> 42 % of mapped reads might seem problematic. Since the sequenced plasmid was extracted from E.coli, there is a lot of contamination.)

# SAMs to BAMs

samtools view -bS ../paired.SAM > paired.BAM
samtools index paired.BAM

# BamCoverage
BamCoverage -b paired.BAM -o BamCoverage/

This does not work and results in the following error message:

"Samtools reports that the number of mapped reads is zero for the file paired.BAM. Please check that the file is properly indexed and that it contains mapped reads."

When i google for this error message i cannot find a helpful answer. Maybe someone knows what is wrong. Since 1979307 reads could be mapped, i don't get why zero reads should be mapped.
Thanks a lot, Alex
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