Error With Bam file Creation

[shwetaJones]

We ran bwa-meth method, following the traditional method where we are converting a sam into a bam file using SortSam with picard tools and am encountering the following errors. Could you please advise how to troubleshoot this?

----- Error log ---- /SNU-719/SNU-719.sam; Line 12828 Line: M04185:48:000000000-DN265:1:1102:15794:5048:ACTAGGAG+GATCAAGT 77 0 0 * * 0 0 ATAATGATTAGTTTAAAATTTGGGGTTTTTTATAATAAGTAATTTGATACGTTATTAGTTAGTTTTTTAAATGAATAAGTAATTATAATAGAAAGAATTAATAATATTGTTGTTTTGAAAAAGGATGATAAAATAATAGGGG FFFGGGGGGGGGGHHHHHHHHHHGGEFGGGGHHHHHHHHHHHHHHHHHHHGHHHHHHHHHHHHHHHGGHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHGHHHHGGHHHHHHHHHHHHHHHH0F AS:i:0 XS:i:0 RG:Z:SNU-719_S2_R_001 YC:Z:CT RX:Z:ACTAGGAGGATCAAGT Ignoring SAM validation error due to lenient parsing: Error parsing text SAM file. RNAME '' not found in any SQ record; File SNU-719.sam; Line 12828 Line: M04185:48:000000000-DN265:1:1102:15794:5048:ACTAGGAG+GATCAAGT 77 0 0 * * 0 0 ATAATGATTAGTTTAAAATTTGGGGTTTTTTATAATAAGTAATTTGATACGTTATTAGTTAGTTTTTTAAATGAATAAGTAATTATAATAGAAAGAATTAATAATATTGTTGTTTTGAAAAAGGATGATAAAATAATAGGGG FFFGGGGGGGGGGHHHHHHHHHHGGEFGGGGHHHHHHHHHHHHHHHHHHHGHHHHHHHHHHHHHHHGGHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHGHHHHGGHHHHHHHHHHHHHHHH0F AS:i:0 XS:i:0 RG:Z:SNU-719_S2_R_001 YC:Z:CT RX:Z:ACTAGGAGGATCAAGT Ignoring SAM validation error due to lenient parsing: Error parsing text SAM file. POS must be non-zero if RNAME is specified; File SNU-719.sam; Line 12828 Line: M04185:48:000000000-DN265:1:1102:15794:5048:ACTAGGAG+GATCAAGT 77 0 0 * * 0 0 ATAATGATTAGTTTAAAATTTGGGGTTTTTTATAATAAGTAATTTGATACGTTATTAGTTAGTTTTTTAAATGAATAAGTAATTATAATAGAAAGAATTAATAATATTGTTGTTTTGAAAAAGGATGATAAAATAATAGGGG FFFGGGGGGGGGGHHHHHHHHHHGGEFGGGGHHHHHHHHHHHHHHHHHHHGHHHHHHHHHHHHHHHGGHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHGHHHHGGHHHHHHHHHHHHHHHH0F AS:i:0 XS:i:0 RG:Z:SNU-719_S2_R_001 YC:Z:CT RX:Z:ACTAGGAGGATCAAGT Ignoring SAM validation error due to lenient parsing: Error parsing text SAM file. Alignment start should != 0 because reference name != *.; File SNU-719.sam; Line 12828 Line: M04185:48:000000000-DN265:1:1102:15794:5048:ACTAGGAG+GATCAAGT 77 0 0 * * 0 0 ATAATGATTAGTTTAAAATTTGGGGTTTTTTATAATAAGTAATTTGATACGTTATTAGTTAGTTTTTTAAATGAATAAGTAATTATAATAGAAAGAATTAATAATATTGTTGTTTTGAAAAAGGATGATAAAATAATAGGGG FFFGGGGGGGGGGHHHHHHHHHHGGEFGGGGHHHHHHHHHHHHHHHHHHHGHHHHHHHHHHHHHHHGGHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHGHHHHGGHHHHHHHHHHHHHHHH0F AS:i:0 XS:i:0 RG:Z:SNU-719_S2_R_001 YC:Z:CT RX:Z:ACTAGGAGGATCAAGT Ignoring SAM validation error due to lenient parsing: Error parsing text SAM file. Reference sequence not found in sequence dictionary.; Line: M04185:48:000000000-DN265:1:1102:15794:5048:ACTAGGAG+GATCAAGT 77 0 0 * * 0 0 ATAATGATTAGTTTAAAATTTGGGGTTTTTTATAATAAGTAATTTGATACGTTATTAGTTAGTTTTTTAAATGAATAAGTAATTATAATAGAAAGAATTAATAATATTGTTGTTTTGAAAAAGGATGATAAAATAATAGGGG FFFGGGGGGGGGGHHHHHHHHHHGGEFGGGGHHHHHHHHHHHHHHHHHHHGHHHHHHHHHHHHHHHGGHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHGHHHHGGHHHHHHHHHHHHHHHH0F AS:i:0 XS:i:0 RG:Z:SNU-719_S2_R_001 YC:Z:CT RX:Z:ACTAGGAGGATCAAGT

[brentp]

hi, did you use bwa mem via bwa-meth? or bwa-mem2 or other? if you can make a pair of fastqs with just these reads, I could try to have a look.

[shwetaJones]

I ran bwameth.py and provided a reference and 2 fastq.gz files +which is using bwa-mem via bwa-mem via bwa-meth. What is the difference between bwa mem via bwa-meth and bwa-mem2 or other? I have the fastq.gz files along with its corresponding sam file, how would you like me to send it to you?

[shwetaJones]

Please let me know where I can send you the test.fastq.gz files so you could have a look.

[brentp]

you can paste t hem here, or just email them to [email protected], should be 1 read, 4 lines, in each file

[shwetaJones]

I sent it over to your email!