Problem somewhere during paired-end seq analysis -> no reads mapped

[Alex852013]

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_index="path/6008.5_BAC.ebwt"
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/paired.bw

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

[Alex852013]

I got it on my own, after a short break. I forgot to sort the bam files...

[colindaven]

One point - it looks like you are using bowtie for alignment, which is obsolete for most purposes. Bowtie2 is a much more accurate aligner, and can detect indels. BWA is another great alternative, as is (commercial) Novoalign.

[Alex852013]

Thanks for the hint. My adviser told me it is sufficient to use Bowtie 1.
I tested Bowtie 2 now and i got 10 % more alligned reads. For another sample even 23 %, which is really a lot!
Thanks again!

[westerman]

Let's not forget BBMap as a good non-commercial alternative.

[GenoMax]

@Alex852013: A large % of reads is still failing to align (if you are only getting 23% of reads to align) so you may want to investigate why that is so. Quick blasting @NCBI should give a clue as to whether you data has unexpected contamination.

[westerman]

@Alex852013: A large % of reads is still failing to align (if you are only getting 23% of reads to align) so you may want to investigate why that is so. Quick blasting @NCBI should give a clue as to whether you data has unexpected contamination.

Didn't he say 23% more not 23% total? In any case the original post talked about mapping a mixture of plasmid and E.coli reads against the plasmid and thus he expected a low percentage.

[GenoMax]

I missed that fact from the original post. @Rick: Thanks for pointing that out. If true #6 can be safely ignored.

[Alex852013]

Yes, it meant 23 % more, which makes 96 % in total. Perfect!
Nevertheless, thanks for caring!