Trimmed NGS exome data failed to be mapped with bwa

Question

[yanqiangchang]

Hi guys I am trying to do some QCs with my human target regions capture data. But After be trimmed with Btrim64, I found that the data can not mapped to the reference genome.

Commands I used are followed:

btrim64 -a 20 -w 5 -l 50 -q -t BKA2/rawdata/305.R1.fastq -o BKA2/map2/305.R1.tr.fqstq 

btrim64 -a 20 -w 5 -l 50 -q -t BKA2/rawdata/305.R2.fastq -o BKA2/map2/305.R2.tr.fqstq 

bwa aln -n 0.04 /BWA_index/hg19.fasta 305.R1.tr.fastq > 305.R1.tr.sai

bwa aln -n 0.04 /BWA_index/hg19.fasta 305.R2.tr.fastq > 305.R2.tr.sai

bwa sampe /BWA_index/hg19.fasta 305.R1.tr.sai 305.R2.tr.sai 305.R1.tr.fastq 305.R2.tr.fastq > 305.tr.sam

Alignment result:

@PG ID:bwa  PN:bwa  VN:0.7.12-r1039 CL:bwa sampe /BWA_index/hg19.fasta 305.R1..tr.sai 305.R2.tr.sai 305.R1.tr.fastq 305.R2.tr.fastq

E00591:39:HKVC5ALXX:7:1101:3671:1520    99  chr9    83467848    60  92M =   83468256    500 CCCNGGAGGCGGAGCTGTGGTGAGCTGAGATTGCGCCNTTGCACTCCAGCCTGGGCAACAAGAGCGAAACTCCAGCTCAAAAAAAAAAAAAA    -AF#F-FJJJJJJAFAFJJJJJJJJJJJJJJJJFF<J#JJJJJJJJJJJJFJ<J<FJJJJFFJFJFJAFJFJJ7-7<FJJJJJJJ<FJJJJJ    XT:A:U  NM:i:3  SM:i:37 AM:i:37 X0:i:1  X1:i:0  XM:i:3  XO:i:0  XG:i:0  MD:Z:3T33T36T17

E00591:39:HKVC5ALXX:7:1101:3671:1520    147 chr9    83468256    60  92M =   83467848    -500    GATTATTTATTTTAGGTAAATTATGGTTCAGTACAATTGATTTTTTAAAAAAAGCGTTTCCCAAAGTGATCAACGTGGAGGGCAGGAAACAG    -A<FJJJFFFFJJFFFFFJA<JJJJAAF<JAFA7FA<FFFFJJJFJJJFJFF-J<JAF<7<7JJJFFFF7FJA-F<F7AFA<JJ<JJFF<AA    XT:A:U  NM:i:2  SM:i:37 AM:i:37 X0:i:1  X1:i:0  XM:i:2  XO:i:0  XG:i:0  MD:Z:2G70A18

E00591:39:HKVC5ALXX:7:1101:5223:1538    65  chr4    13325926    37  116M    chr14   32605360    0   TGCNTAGGTTCCTGCTCACTTTCCAATTCTTAATTCCAGTCCCTTCAGAGCCCCACTGCTCCTGGATTCCAAGAACTACCTAAGTGTCTTTTTAACAATGACCCATTTTTGCTTAG    <AA#F7FFFAJJJFFJJJFJJJJJAFJ<7FJFJFJJJJJJJJJJJFJFJFFFJJFJJFJFJJJJAFFJJJJJAJ-FJFJJJJ<JJJ7JA-7JJFAJ<FJ7AJJFFFFJJJFJJJA-    XT:A:U  NM:i:2  SM:i:37 AM:i:37 X0:i:1  X1:i:0  XM:i:2  XO:i:0  XG:i:0  MD:Z:3C111A0

E00591:39:HKVC5ALXX:7:1101:5589:1538    129 chr14   32605360    37  96M chr4    13325926    0   GTTAACCATTTTAAAGTATATAATTCAGAGGCATTTAGTATATTCACAATGTTGTGCAACCATCACCTCTGGTTGCAAAATATTTTAATGGCTCCA    <AAAFJJJJJ<JJJJA-FJJJJJJJJJFJF<JJFAJJAFJJJJJJJJJJJ-FJFJ<JJJJFJJJJJJJJJ<7FF-<FJFJJFJJFJ<AF<-AJJJ)    XT:A:U  NM:i:1  SM:i:37 AM:i:37 X0:i:1  X1:i:0  XM:i:1  XO:i:0  XG:i:0  MD:Z:89T6

Only 4 sequence were aligned to the ref.

I have never experienced things like that. Dose anybody know why this things happened ?

PS: Without trimming, reads can be mapped normally to the ref.

[finswimmer]

Hello,

how many reads survive your trimming?

fin swimmer

[yanqiangchang]

3q4 ur reply. For R1 9290975 reads out of 9540037 passed the filter, for R2 7863295 reads out of 9540037 passed the filter. 7784615 reads was paired.

[WouterDeCoster]

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Answer 1

[yanqiangchang]

Solved. After data trimming, the R1 and R2 files must be sorted.