Trying to follow the following recommended procedure ""http://www.nimblegen.com/products/lit/07187009001_NG_SeqCap_TechNote_EvalEZdata.pdf"
Have seen a lot of similar posts on bioconductor about sam headers, unfortunately none that sheds light on this problem.
Anyone have a clue?
Thanks, Chris
Background:
>>> java -jar ../apps/trimmomatic/trimmomatic-0.32.jar PE -phred33 sample_R1_001.fastq sample_R2_001.fastq sample_R1_001_trimmed.fq sample_R1_001_unpaired.fq sample_R2_001_trimmed.fq sample_R2_001_unpaired.fq ILLUMINACLIP:../apps/trimmomatic/adapters/TruSeq3-PE.fa:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:5:20 MINLEN:75
TrimmomaticPE: Started with arguments: -phred33 sample_R1_001.fastq sample_R2_001.fastq sample_R1_001_trimmed.fq sample_R1_001_unpaired.fq sample_R2_001_trimmed.fq sample_R2_001_unpaired.fq ILLUMINACLIP:../apps/trimmomatic/adapters/TruSeq3-PE.fa:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:5:20 MINLEN:75
Multiple cores found: Using 2 threads
Using PrefixPair: 'TACACTCTTTCCCTACACGACGCTCTTCCGATCT' and 'GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT'
ILLUMINACLIP: Using 1 prefix pairs, 0 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences
Input Read Pairs: 65290734 Both Surviving: 45006507 (68.93%) Forward Only Surviving: 12263552 (18.78%) Reverse Only Surviving: 2683491 (4.11%) Dropped: 5337184 (8.17%)
TrimmomaticPE: Completed successfully
>>> /path/to/bwa mem -R "@RG\tID:TEST_SAMPLE\tPL:illumina\tLB:TEST_SAMPLE\tSM:TEST_SAMPLE" /path/to/ref.fa –t 4 -M SAMPLE_R1_trimmed.fq SAMPLE_R2_trimmed.fq | /path/to/samtools view -Sb - > SAMPLE_unsorted.bam
>>>head -n1 Sample_unsorted.bam gives
HWI-ST1337:134:C41YMACXX:8:1101:1239:1466 99 chr10 46233512 55 101M = 46233685 274 TCTTAGCTGAGTTTCTGACTTTGAAAAGTGTGGTGGAGATCGATGTGTTATGTGGGAACTGGGGGATGGTGGGTGGGGTTGGGAAAGGGAGGGACTGCTCC @CCFFFFFFFHHHIHIIGGIJJIBHHIGEHCHHFHGFHGHIGGEIHIGHHIIIIDHBFGFHIIJJBBBD;A?B9>BBB<9BBD+388ABABDD@8?<AA@: NM:i:0 MD:Z:101 AS:i:101 XS:i:91 RG:Z:TEST_SAMPLE XA:Z:chr10,+47894653,101M,2;chr10,+51838516,101M,2;
>>> ../samtools sort SAMPLE_unsorted.bam SAMPLE_sorted
output:
[E::sam_parse1] missing SAM header
[W::sam_read1] parse error at line 1
[bam_sort_core] truncated file. Continue anyway.
Info:
bwa-0.7.10
picard-tools-1.121
samtools-1.1
Trimmomatic-0.32