bwa mem: paired end reads have different names: Issue with 0.7.10

Question

[ARB]

Hi all,

I am trying to run alignments for paired end MiSeq data and I am getting this error:

[mem_sam_pe] [mem_sam_pe] [mem_sam_pe] paired reads have different names: "M03721:10:000000000-AH7UG:1:1101:16268:1596", "M03721:10:000000000-AH7UG:1:1101:16959:1596"

paired reads have different names: "M03721:10:000000000-AH7UG:1:1101:13391:1606", "M03721:10:000000000-AH7UG:1:1101:17720:1609"

[mem_sam_pe] paired reads have different names: "M03721:10:000000000-AH7UG:1:1101:16099:1561", "M03721:10:000000000-AH7UG:1:1101:17123:1563"
[mem_sam_pe]

I tried sorting my fastq, also ran the repair.sh tool from BBtools, but nothing seems to work.

Here is the command I am using:

~/bin/0.7.10/bwa mem -M -v 1 -t 24 -R $readGroup -p $fasta 1.fastq 2.fastq 1> raw.sam 2> Logs_bwaAlign.txt

I am using 0.7.10 bwa for alignment. However, when I use an older version such as 0.7.5a or newwer version like 0.7.12, I do not get this error and alignments works fine. This is only the issue with 0.7.10 and it really baffles me. I cant seem to figure what is the issue here. If you can help me understand whats going on here, that would be great.

Thank you so much in advance.

Ashini

Here are the first few reads from my fastq (that are giving issues) for anyone to look. I cant seem to find an attachment option, so pasting it here.

1.fastq:

@M03721:10:000000000-AH7UG:1:1101:15914:1524 1:N:0:3
TTTGTGTGCTGGGTTACAGAGCCACACCACTCATCCCTAGCCTTGTCATGTTAGCTATCCATTCTGTCTAACGTAGTGGCTTATCCCAAACCTTAGTGGTTTAAAACTTATCCCCAAAGTTAATGTGGTTTAAAAGAACAACATGCTGGCC
+
>>A1A1C1DFB11FF1B3111BF0EFEEAFHHBFGGGFB0EEGD1GFFGFGHB1AF2D0F2FEGHGHFH22F/B00F00AGFFHFE@100??FG11BF0GFE@2111FF1FGFFC0/00BF22BBB21BFG12111/0<B0//B11FB1<<
@M03721:10:000000000-AH7UG:1:1101:17949:1533 1:N:0:3
TGTAATACACGGCCGTGTCCTCAGATCTCAGGCTGCTCAGCTCCATGTAGGCTGTGCTCGTGGATTTGTCCGCGGTAATCGTGACTCTGCCCTGGAACTTCTGTGCGTAGTTTGCTATACCAAGGATAGGGATGATCCTTCCCATCCACTT
+
11>11D3D1C11AA0E0FFFGF111DCFF220AF0FGDB0BFFF1F1FB21BFCG0AF1/EA//BGG1DFF/>//>//BF/F00?FBF1@EEG0/01BGHDE1B21/?//FEFB>F2B2BF110/0B11000B/1@GFGHFFF1@FC1FF1
@M03721:10:000000000-AH7UG:1:1101:17725:1536 1:N:0:3
TAGTTCGGGCCCTGGGGTCTTGCTGGGGCCCATAGGCATGTGGGGCAGGAAGCTGGGCAGAGCTGGCCTGGCTCTGAGTGGCCACTGGGGGAAAGGCAGCCTTCTGTCTGCCAATCTGGGTAGGCCCAGTGGCCAAGCTCTCATGGGAGAG
+
>11>AD111>A@GA000AEEG1AF1100AAA/G011E1FAFE1//A/A//00AF10/E0///>F10?BG//BGEGB11G1/>F1?F10//////00?0??F0FC<1BBF1>B11@FG210@1//1@?//?10?<1//<><=<1=11.0>..
@M03721:10:000000000-AH7UG:1:1101:17080:1536 1:N:0:3
TTTTTGTCTTTTGTTGTTGTTCAAACGGGATTCACAGAGTATTTGAAAAATGTATATATATTAAGAGGTCACGGGGGCTAATTGCTGGCTGGCTGCCTTTTGCTGTGGGGTTTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
+
1>AAA1CDFFFFCGGCGGAFGEB33FA?C0BGG1D211AF2DGHB21110BEF2F2FEF2GG22A11/FG1B/////EEC1BG21GB1EF//?FCEGGHHHBDFDF0B/?ECEF201BF/////00@1<0</??G/1<1FF<11?<11>G1
@M03721:10:000000000-AH7UG:1:1101:13934:1558 1:N:0:3
TAGCTGCTCCCCAGACAAGCAGTGCATGTGAGCAGCTGGGATGCCACAGGAGGGAGGTTTCTTTTGGGGGCTGTACCACTGTGGGAGGAGTTTGTAGAGCTTGCATGCAGTGATAAACCCCAACATCTTCAGCCTCCACCCGGCTNNNNNN
+
111>A1CCDFAAB?1FB11EC0FBFBGBGBDEFDBFGGB?AGCGGFGF0A0/A?/A?EGHHHHHH1////EE?GEGHBGHFHGFC/C///GHHBHFD2DEGGBE1GBG1DGB1F221BCECCCC/FGHHG21@FHHHBFGF//<A/01FF.
@M03721:10:000000000-AH7UG:1:1101:17966:1558 1:N:0:3
TTTTCTTTGTCTACAATATGGGAAGAGCCCTGGTTTTGCCTGCTTCCAGGGCTATTGTGAGATCAAAGGAAAGAATGCAAGTGCAAGAGTTTTGAGGACAGCAACATCCTATTTGGCTTTGAATTATTAATATGACTTGCTTAAAAGTTTA
+
1>AAAFFF3FFF3B11F1F31111A000BFH00FGHF0AFHBGGHHFE10/BF0BG1G2211BFCB11B00B00BB1F111FD1A1100DGGH0//01B0>/0000BFGHDGHG21@GEG121BF1FD22B2B>2BFG11BB21111FGH2
@M03721:10:000000000-AH7UG:1:1101:16099:1561 1:N:0:3
TAATAAAAAATATGTATATAATAAATTATTGTTGACTATGGTCACCCTGTTATGCTAATACTAGATCTTATTCACTCTATATTTTTGCACTCATTAACCATCTATACTTTTTCCCCCATCCCCACTACCCTTCCCAGTCTCTGGNNNNNNN
+
111>1@D111C1GBG3F3FDFGB31DGBGHDHHFBGHCH21FGCHGGHFHHEHEFHBFHFFG2A1FFGH2FGD2DHGHGHFHHHHH/12ABGBGHFEEF1F@G1F2FGHHHHHFGFE?GGCGF0EG0FCCEGHF011FBGHH11101FGA/
@M03721:10:000000000-AH7UG:1:1101:17123:1563 1:N:0:3
TACCATGTTCAATGATTTCAACTAAACTTCTAAGATGTGGCAAGATGGCACAGCCCATAAGAATAGCTATCTGTTGTACAATCTTAATACCAGTGTGTCTCGCTTGCCAGGACTTCTTGCTTTTGCACACAGCTTTTAAGAAGGGCAATAA
+
>1>>>DDFFFBFGG1GGGG3DGHFB1FGHFHFBEFGBG11A0100GFFGFF00FGE/F01111FAFFGBFGH2GG1FFGHEHHHHEBGBGGE1FCFFHHHD0FFHFGEF1//?FHGHHBFHHHH1DBF1F/0?GHHHE111100/</<G11

2.fastq:

@M03721:10:000000000-AH7UG:1:1101:15914:1524 2:N:0:3
CTCGGCCTCCCAATGTGCTGGGTCTACAGGCATGAGCCACCATGCCTTGCCAGCATGTTGTTCTTTTAAACCACTTTCACTTTGGGGCTAAGTTTTAAACCACTAAGGTTTGGGATAAGCCACTACGTTAGACAGACTTGATAGCTAACAT
+
1>>1111>11>1111B33B11000B1A11000B011A00AB0B1A1A11BA11/0B1DF2BF1FGHH211AA/A11B12BDFG10///>011BFGH21@10/>B0110BFG////B0111>0>B00/BF0/111000B211>22>B2101B
@M03721:10:000000000-AH7UG:1:1101:17949:1533 2:N:0:3
TGTTGTTGGTTTGGATCATCCCTCTCCTTGGTATAGCTATCTACTCACTGAAGTTCCAGGTCTGAGTCACGTTTACCGCGGTCTAATCCACTAGCACAGCCTACATGGAGCTGTGCTGCCTTAGATCTTTGGTCCCTGCCGTGTCTTACTT
+
11>111>11111111BA1B3AAA0BA0BG11B1D221222AD21211111111DG2A2101B1011D111/00B/11///////12B@B1@10101100/>B1B1@2010>>110121BB>1111B>B1210/11011/</?/B0BB21>2
@M03721:10:000000000-AH7UG:1:1101:17725:1536 2:N:0:3
CTCCCATTATTGCTTTGCCACTTGGCCTACCCAGATTGGCAGACAGACGTCTGCCTTTCCCCCAGTGGCCACTCAGATCCATGCCAGCTCTTCCCAGCTTCCTGCCCCACATGCCTTTGGGCCCCCGCTAGACCCCTGGCCCCGAACTCAT
+
1>>>>1B133333BF31111BB1111AB0BBA100BF10A100A0000//AA/1AFFG1AAAB//B10/A1AB00112@11211111/B@F1@B>110BF11B1@B/?/>/B1B1>1B10/0/<///<///111??///00?<///>?111
@M03721:10:000000000-AH7UG:1:1101:17080:1536 2:N:0:3
TTTACCCCTCTTCAACATGCAGCCTGCCCGCAATTAGCCCCCGTTCCCTCTTAATATATATACATTTTTCAAATTCTCTGTTAATCCCGTTTGAACAACAACACAAGACCACACAGTTCGGCATTGCTTCGTTTTGGGATTGTGTGTATAT
+
1111>1111>11A3131111111A000B00000BB110AAAA/A00AABAFA12A2D2D2D2B2DADAG0222D1@@2D1B222D2@B/BBF/11@10////////00>00//>/01>2/////1112>1/<0?/0///10012<0<2222
@M03721:10:000000000-AH7UG:1:1101:13934:1558 2:N:0:3
CGCCGGGTGGAGGCTGAAGATGTTGGGGTTTATCACTGCATGCAAGCTCTACAAACTCCTCCCACAGTGGTACAGCCCCCATAAGAAACCTCCCTCCTGTGGCATCCCAGCTGCTCACATGCACTGCTTGTCTGGGGAGCAGCTCNNNNNN
+
11>>111>10000AA00BA1B1DD200AEEEFHF2FG1A1D2B111ABBG1B111AFCGGFFE0B/0B10D2B12B>??//0011110BBGE?EGFEH1B10?0B>FC11<B11BF1<1B1<1BF0BFF1BFF100///</0??F101??/
@M03721:10:000000000-AH7UG:1:1101:17966:1558 2:N:0:3
TCCTAGCACATCTTGGATTAGTCTCGTAATAAACTTTTATGCTCGTCATATTAATAATTCAAAGCCAAATAGTATTTTTCTGTCCTCTAATCTCTTGCACTTGCATTCTTTCCTTTGATCTCTCACTTTCCCTGGAATCATGCAAAACCAT
+
111>11@111113B311BB11B3B30A00B33A1BGFF2121210AA0A2DB22B22ADB122100B00A1122DDAF1AD2AAAFA11121BDEG1111BF111BGFEEGD@GGF12@@F22B11B1111B@1/0111121111100?/1
@M03721:10:000000000-AH7UG:1:1101:16099:1561 2:N:0:3
CCAGAGACTGGGAAGGGTAGTGGGGATGGGGGAAAAAGTATAGATGGTTAATGAGTGCAAAAATATAGAGTGAATAAGATCTAGTATTAGCATAACAGGGTGACCATAGTCAACAATAATTTATTATATACATATTTTTTATTTNNNNNNN
+
1>11111>B11111111F10B00000B0000/A//00/B2D222D21BGB1GB2AFDDF110/B1F2221F112D2111DBG11F2GHD2B@GB@FB1//FA/BFEG11GGB2B1/B>1FGH2FG2GBG2>2F2FHHHGGEHHHB21B>//
@M03721:10:000000000-AH7UG:1:1101:17123:1563 2:N:0:3
CTGGATGAGTATGTCCGTAACACAACAGCTAGAGCTTTTGCTGTTGTATCCTCTGCCCTGTGCATTCCTTCTTTATTGCCCTTCTTAAAAGCTGTGTGCAAAAGCAAGAAGTCCTGTCAAGCGAGACACACTGGTCTTAAGATTGTACAAC
+
1>111B111B3@3BAB1F11A1B00A00BFAF111BGHH11F1DF1B12D2FDG11FFF01112DGFAAEDGHH2FG1AABFF0FG22111>F1D0F0D21100>100000BB@G11@221@////>0B0?B10B1BF2211BF1B2>211

Answer 1

[SES]

Try the command without the -p or interleave the pairs (the -p says to ignore the 2.fastq file). Also, you can just post one or a couple of records next time instead.

[ARB]

I am already using the -p flag in my alignment.

~/bin/0.7.10/bwa mem -M -v 1 -t 24 -R $readGroup -p $fasta 1.fastq 2.fastq 1> raw.sam 2> Logs_bwaAlign.txt

I apologize for pasting so many records. I will keep that in mind next time.

Thanks

[h.mon]

SES said to NOT use -p. This option is for "smart pairing":

-p
Smart pairing. If two adjacent reads have the same name, they are considered to form a read pair. This way, paired-end and single-end reads can be mixed in a single FASTA/Q stream

As you have proper paired files, you do not want to use -p.

[ARB]

Oops, my bad. I misread it. I removed it and it worked. Thank you so much SES and h.mon.