[mem_sam_pe] paired reads have different names. Without -p

Question

[windsur]

Hello, I am newbi in processing fasq files, but it is the first time that happened to me. I downloaded the L00N_RN_00N.fastq.gz, and joined in two fastq files (i.e. _R1.fastq.gz and _R2.fastq.gz). Then I load the genome reference to memory and I make a loop samples for BWA, but today I have an error message:

> [mem_sam_pe] paired reads have different names

And this is my code that I used.

call('bwa mem -t' + str(args.threads) + ' -R "@RG\tID:' + sample_name + '\tLB:library\tPL:illumina\tPU:library\tSM:' + sample_name + '" ' + genome_ref + ' ' + forward_paths[i] + ' ' + reverse_paths[i] + ' > ' + sample_path + '/' + sample_name + '_bwa.sam',shell = True)

I am quite confuse, because I have analyse several samples as always but I've never seen that. Any help is welcome! :) I downloaded the files again and joined them.

[windsur]

Nice! I'm going to check it right now. Thanks! If it is "0", what should I do?

Answer 1

[DareDevil]

I had similar kind of error. I tried following to fix that error:

gunzip -c sample.fastq.gz | sed -E 's/(^[@+]SRR[0-9]+\.[0-9]+)\.[12]/\1/' | gzip -c > sample.fixed.fastq.gz

Answer 2

[Pierre Lindenbaum]

its's not a problem with bwa, it's a problem with

 forward_paths[i] + ' ' + reverse_paths[i]

your fastq files are not the correct pair R1/R2 or the sort order of the read is not the same between R1 and R2, or there are some empty reads in your fastqs

test:

gunzip -c name.R1.fastq.gz | paste - - - - | cut -f 1| head
gunzip -c name.R2.fastq.gz | paste - - - - | cut -f 1| head

and

gunzip -c name.R1.fastq.gz name.R2.fastq.gz  | paste - - - - | cut -f 2| grep  -E '^$' | wc -l

must be '0'

[windsur]

Thank you! I just checked: For the first one (R1):

@NS500387:143:HVKMFAFXX:1:11101:14111:1058 1:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:6775:1061 1:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:21397:1063 1:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:11426:1064 1:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:10515:1066 1:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:3391:1066 1:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:11290:1066 1:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:5843:1071 1:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:8819:1077 1:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:6162:1078 1:N:0:TAGGCATG+NTCCTTAC

and (R2):

@NS500387:143:HVKMFAFXX:1:11101:14111:1058 2:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:6775:1061 2:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:21397:1063 2:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:11426:1064 2:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:10515:1066 2:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:3391:1066 2:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:11290:1066 2:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:5843:1071 2:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:8819:1077 2:N:0:TAGGCATG+NTCCTTAC
@NS500387:143:HVKMFAFXX:1:11101:6162:1078 2:N:0:TAGGCATG+NTCCTTAC

the last one return me the value of "0".

[windsur]

@PierreLindenbaum this is the message that I get:

[mem_sam_pe] paired reads have different names: "NS500387:143:HVKMFAFXX:1:11101:21856:1052", "NS500387:143:HVKMFAFXX:2:11101:12190:1028"

[weiqiao1029]

Hi @PierreLindenbaum Sorry to disturb you for this old question. I got some fastq from my PI, then I fastqc them showing no adapter.I am not sure how they library and trim, maybe so do my PI. Besides, when I bwa-men without -p it shows [mem_sam_pe] paired reads have different names then I checked them:

$ gunzip -c E1_input.fq.gz | paste - - - - | cut -f 1| head
@CL100056099L2C001R002_24
@CL100056099L2C001R002_40
@CL100056099L2C001R002_45
@CL100056099L2C001R002_73
@CL100056099L2C001R002_74
@CL100056099L2C001R002_81
@CL100056099L2C001R002_90
@CL100056099L2C001R002_91
@CL100056099L2C001R002_95
@CL100056099L2C001R002_103
$ gunzip -c E1_pulldown.fq.gz | paste - - - - | cut -f 1| head
@CL100056099L2C001R002_61
@CL100056099L2C001R002_115
@CL100056099L2C001R002_154
@CL100056099L2C001R002_218
@CL100056099L2C001R002_228
@CL100056099L2C001R002_253
@CL100056099L2C001R002_255
@CL100056099L2C001R002_269
@CL100056099L2C001R044_184179
@CL100056099L2C001R002_305
$ gunzip -c E1_input.fq.gz E1_pulldown.fq.gz  | paste - - - - | cut -f 2| grep  -E '^$' | wc -l
0

Then I bwa-men with -p it succeed. I am struggling about why there are not the correct R1/R2 or I am not sure whether they are really paired-end fastq. And in the following steps I would like to samtools to sorted bam then ATACseqQC. Cause it become one E1.sam after bwa-men with -p I am not sure whether it will affect the following results.or I just bwa aln for single-end reads?or what is the difference between bwa-men with -p for paired-end and bwa-aln for single-end?Thank you so much for this stupid question. Could you please give me some advice about my following analysis?Thank you so much in advance.

[DareDevil]

gunzip -c sample.fastq.gz | sed -E 's/(^[@+]SRR[0-9]+\.[0-9]+)\.[12]/\1/' | gzip -c > sample.fixed.fastq.gz

Answer 3

[windsur]

Solved! It wasn't a problem of my script. The problem was when I've tried downloading the files through Basespace of Illumina, for some reason, there was a connection problem and the files was "corrupted". what I did is downloading the files and then join the fastq files (separated).