Hi Fang, I think it might be that you need to include "h" in your call to samtools view to include the header in the binary you're piping to samtools sort (I.e., samtools view –bSh SMDC…). Equivalently, you could use the –t option with view and pass the index of the reference FASTA file.
And as an aside, for RNA-seq reads, you might be better off aligning with an intron-aware aligner like STAR, bowtie/TopHat, or GSNAP. Bwa-mem was written to align DNA sequence reads. Best, --Nancy -- ************************************* Nancy F. Hansen, PhD [email protected] Comparative Genomics Analysis Unit, NHGRI 5625 Fishers Lane Rockville, MD 20852 Phone: (301) 435-1560 Fax: (301) 435-6170 From: 刘放 Fang Liu <[email protected]<mailto:[email protected]>> Date: Thursday, June 11, 2015 12:40 AM To: "[email protected]<mailto:[email protected]>" <[email protected]<mailto:[email protected]>> Subject: [Samtools-help] wrong output format of bwa? Dear all, I just started analyzing my RNA-seq results, first, I used command bwa mem -t 20 transmycale95300.fasta SMDC-1_R1_shortReadRemoved.fq SMDC-1_R2_shortReadRemoved.fq > SMDC-1_aln-pe.sam to generate sam files for downstream analysis. But then when I ran samtools, it says there’s something wrong with my sam file. samtools view -bS SMDC-1_aln-pe.sam | samtools sort -m 30000000000 - SMDC-1_sorted [bam_header_read] EOF marker is absent. The input is probably truncated. [main_samview] fail to open "SMDC-1_aln-pe.sam" for reading. [bam_header_read] invalid BAM binary header (this is not a BAM file). Segmentation fault (core dumped) And this is the tail output of my sam file: HISEQ:184:C6KGFANXX:1:2316:19939:10050683 fang_sponge_transABySS_contig_1301343|m.1302716-fang_sponge_transABySS_contig_1301343|g.1302716--ORF-fang_sponge_transABySS_contig_1301343|g.1302716-fang_sponge_transABySS_contig_1301343|m.1302716-type_complete-len_1065-(+)-fang_sponge_transABySS_contig_1301343_213-3407(+)2338 6020M2D47M =2238 -169TACCATGTTACTGAAGAAACGTGGTAGAGGAAATGCAGAGTATGCTTTGAAAGTCATAAAGTGTGACFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFFFFBFFFFF<F<BBBBBNM:i:2 MD:Z:20^AT47AS:i:59 XS:i:39 HISEQ:184:C6KGFANXX:1:2316:19939:100506163 fang_sponge_transABySS_contig_1301343|m.1302716-fang_sponge_transABySS_contig_1301343|g.1302716--ORF-fang_sponge_transABySS_contig_1301343|g.1302716-fang_sponge_transABySS_contig_1301343|m.1302716-type_complete-len_1065-(+)-fang_sponge_transABySS_contig_1301343_213-3407(+)2238 6050M3S =2338 169CCTTCGCCATAAAATTGTTCATGAACTCTCTATAACGTTTGATGACAGAACTTFBBBBBFFFFFBFFFFFFFFFFFFFFFFFFFFFFFFFFFFF<FFFFFFFFFFFNM:i:0 MD:Z:50AS:i:50 XS:i:0 HISEQ:184:C6KGFANXX:1:2316:20405:10050077 *0 0* *0 0CTAAAAAACAAAAATTAAAAAAAATCACAAAAAAATCTCATTATATTTTAAGAAAGTTTATGAATTTGTGTTGGACTAGGGGTTGGACBBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAS:i:0 XS:i:0 HISEQ:184:C6KGFANXX:1:2316:20405:100500141 *0 0* *0 0CTGTAATATTATGTGTAGATTCTAGTATACACAAAGTGGATACATAACAGTTACTATTTTTCTTTCCTCGTCTCTGAGTAAACCAAGCTTGTCCAACCCCTAGTCCAACACAAATTCATAAACTTBBBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAS:i:0 XS:i:0 HISEQ:184:C6KGFANXX:1:2316:20319:100522117 fang_sponge_transABySS_contig_323248|m.303829-fang_sponge_transABySS_contig_323248|g.303829--ORF-fang_sponge_transABySS_contig_323248|g.303829-fang_sponge_transABySS_contig_323248|m.303829-type_5prime_partial-len_110-(+)-fang_sponge_transABySS_contig_323248_3-332(+)119 0* =119 0TTTCTTATTGCCTTTGACTTTGCTTGCCTTTTATTATCTTTGAAAACTTAGCTTTTTTCATTTGCCTTTGACFFFFFFFFBFFFBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFB<FFFFFFFBBBBBAS:i:0 XS:i:0 HISEQ:184:C6KGFANXX:1:2316:20319:100522185 fang_sponge_transABySS_contig_323248|m.303829-fang_sponge_transABySS_contig_323248|g.303829--ORF-fang_sponge_transABySS_contig_323248|g.303829-fang_sponge_transABySS_contig_323248|m.303829-type_5prime_partial-len_110-(+)-fang_sponge_transABySS_contig_323248_3-332(+)119 6065M =119 0CCATGTGTAGCTCATAAGAGTCTTCAGTAATGTCTTCTAAAGCAGCAACAGTAGAGCACTGACTGFFFFFFFFFFFFFFFFFFBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFBBBBBBNM:i:3 MD:Z:15C42G2G3AS:i:53 XS:i:0 HISEQ:184:C6KGFANXX:1:2316:20364:10052383 fang_sponge_transABySS_contig_1286875|m.1267259-fang_sponge_transABySS_contig_1286875|g.1267259--ORF-fang_sponge_transABySS_contig_1286875|g.1267259-fang_sponge_transABySS_contig_1286875|m.1267259-type_3prime_partial-len_474-(+)-fang_sponge_transABySS_contig_1286875_85-1503(+)888 027M =784 -131TATGCTAACAAGTTCAAAAGAAATGGCFFFFFFFFFFFFFFFFFFFFFFBBBBBNM:i:0 MD:Z:27AS:i:27 XS:i:27XA:Z:fang_sponge_transABySS_contig_1107776|m.1087146-fang_sponge_transABySS_contig_1107776|g.1087146--ORF-fang_sponge_transABySS_contig_1107776|g.1087146-fang_sponge_transABySS_contig_1107776|m.1087146-type_3prime_partial-len_584-(-)-fang_sponge_transABySS_contig_1107776_3-1751(-),-888,27M,0; HISEQ:184:C6KGFANXX:1:2316:20364:100523163 fang_sponge_transABySS_contig_1286875|m.1267259-fang_sponge_transABySS_contig_1286875|g.1267259--ORF-fang_sponge_transABySS_contig_1286875|g.1267259-fang_sponge_transABySS_contig_1286875|m.1267259-type_3prime_partial-len_474-(+)-fang_sponge_transABySS_contig_1286875_85-1503(+)784 0122M =888 131CACAAAGTGCAGTTGACTTCACAATCATCAAGTGATGAGAATTCAAGCGATAGTGCAACTACAGGAACCAAATCATCCTCAGACTTATTAGCCTCTACTCCATCTATGCTAACAAGTTCAAAFBBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFNM:i:1 MD:Z:0A121AS:i:121 XS:i:121XA:Z:fang_sponge_transABySS_contig_1107776|m.1087146-fang_sponge_transABySS_contig_1107776|g.1087146--ORF-fang_sponge_transABySS_contig_1107776|g.1087146-fang_sponge_transABySS_contig_1107776|m.1087146-type_3prime_partial-len_584-(-)-fang_sponge_transABySS_contig_1107776_3-1751(-),+784,122M,1; HISEQ:184:C6KGFANXX:1:2316:20580:10050077 *0 0* *0 0TAGACATCGTAGTTGGTCTGTGCATATATGGAACACACCTGTACCAGAGTGAGAAGAAGAGAACTACTTAGAGATTGTTGGTGACATTBBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAS:i:0 XS:i:0 HISEQ:184:C6KGFANXX:1:2316:20580:100500141 *0 0* *0 0CGCTTGGTTTTTCAGTCCACTTCCTGCACTCTTAGTTTAGTGCTTAGAGATAGCACCAATGATCCAATGTCACCAACAATCTCTAAGTAGTTCTCTTCTTCTCACTCTGGTACAGGTGTGTTCCFBBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAS:i:0 XS:i:0 Can anyone tell me how to solve this? Many thanks! Kind. Fang
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