Bowtie2 and BWA-MEM not working

Question

[Matt01]

Hi, so I have been using Galaxy for genome alignment on two sequences (one forward and other reverse) obtained from Illumina sequencing. The sequences are DNA from exome sequencing. I have realized that when I use the tool "filter quality by data" on my two sequences and then try to align them using either Bowtie2 or BWA-MEM, I get an error message. The error message I get from Bowtie 2 is:

Fatal error: Exit code 1 ()
Tool generated the following standard error:

[bam_sort_core] merging from 16 files and 8 in-memory blocks...

The error I get from BWA-MEM is:

[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] skip orientation FR as there are not enough pairs
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
[mem_sam_pe] paired reads have different names: "K00171:532:HLKHHBBXX:5:1101:29254:1754", "K00171:532:HLKHHBBXX:5:1101:29721:1754"

[mem_sam_pe] [mem_sam_pe] paired reads have different names: "K00171:532:HLKHHBBXX:5:1101:28848:1754", "K00171:532:HLKHHBBXX:5:1101:29315:1754"

[mem_sam_pe] paired reads have different names: "K00171:532:HLKHHBBXX:5:1101:28645:1754", "K00171:532:HLKHHBBXX:5:1101:29011:1754"

Why is it the case? Why is it that when I apply the filter tool on sequences, the alignment tools just give up, whereas when I don't filter sequences, they align perfectly. How do I overcome this?

Kind regards, Matt

[Pierre Lindenbaum]

there is a problem with your fastq files and or the order of the reads in your fastq files . see paired reads have different names (bwa-mem) ; bwa error: paired reads have different names ; etc

for example; you should have the same read names left/right for the following command:

paste  <(gunzip -c in.R1.fq.gz | paste - - - - | cut -f 1)  <(gunzip -c in.R2.fq.gz | paste - - - - | cut -f 1) | grep -F "K00171:532:HLKHHBBXX:5:1101:29254:1754" -m2 -C 3

[GenoMax]

I use the tool "filter quality by data" on my two sequences

Which tool is that in galaxy? Are you processing your read pairs separately?

[Matt01]

No both at the same time and the tool is called "filter by quality"