LeftAlignIndels error

Question

[mailard]

Hello!

I input sorted and indexing bam file to LeftAlignIndels: ~/Soft/gatk-4.1.9.0/gatk LeftAlignIndels -I bam_fin/Exome_dups.bam -R /mnt/lapd/Index_hum/dna2/GRCh_2021.fa -O bam_fin/Exome.bam

And have this error:

'java.lang.IllegalArgumentException: Alignments added out of order in SAMFileWriterImpl.addAlignment for file:///mnt/lapd/Vika_data/RNF_raw/exome/bam_fin/Exome.bam. Sort order is coordinate. Offending records are at [1:152985370] and [1:152985347] at htsjdk.samtools.SAMFileWriterImpl.assertPresorted(SAMFileWriterImpl.java:197) at htsjdk.samtools.SAMFileWriterImpl.addAlignment(SAMFileWriterImpl.java:184) at htsjdk.samtools.AsyncSAMFileWriter.synchronouslyWrite(AsyncSAMFileWriter.java:36) at htsjdk.samtools.AsyncSAMFileWriter.synchronouslyWrite(AsyncSAMFileWriter.java:16) at htsjdk.samtools.util.AbstractAsyncWriter$WriterRunnable.run(AbstractAsyncWriter.java:123) at java.base/java.lang.Thread.run(Thread.java:829)'

I want to note that the unsorted file is the output bam.

I appreciate your help!

With best regards, Viktoriia

[Pierre Lindenbaum]

is Exome_dups.bam sorted and indexed ? what's the output of picard ValidateSamFile with Exome_dups.bam ?

[mailard]

Yes, the file was sorted and indexed.

ValidateSamFile output
 `INFO  2021-11-26 12:06:52 SamFileValidator    Seen many non-increasing record positions. Printing Read-names as well.
INFO    2021-11-26 12:07:42 SamFileValidator    Validated Read    10,000,000 records.  Elapsed time: 00:00:50s.  Time for last 10,000,000:   50s.  Last read position: 16:2,971,202.  Last read name: A00175:147:H3MH3DMXY:1:1231:26973:34992
INFO    2021-11-26 12:08:31 SamFileValidator    Validated Read    20,000,000 records.  Elapsed time: 00:01:38s.  Time for last 10,000,000:   48s.  Last read position: 19:50,278,169.  Last read name: A00175:147:H3MH3DMXY:1:1361:12617:29418
INFO    2021-11-26 12:09:19 SamFileValidator    Validated Read    30,000,000 records.  Elapsed time: 00:02:27s.  Time for last 10,000,000:   48s.  Last read position: 16:31,473,305.  Last read name: A00175:147:H3MH3DMXY:1:2105:11812:14340
INFO    2021-11-26 12:10:08 SamFileValidator    Validated Read    40,000,000 records.  Elapsed time: 00:03:15s.  Time for last 10,000,000:   48s.  Last read position: 10:122,618,152.  Last read name: A00175:147:H3MH3DMXY:1:2256:16034:20588
INFO    2021-11-26 12:10:57 SamFileValidator    Validated Read    50,000,000 records.  Elapsed time: 00:04:04s.  Time for last 10,000,000:   49s.  Last read position: 5:105,805,689.  Last read name: A00175:147:H3MH3DMXY:1:2418:8214:16626
INFO    2021-11-26 12:11:45 SamFileValidator    Validated Read    60,000,000 records.  Elapsed time: 00:04:53s.  Time for last 10,000,000:   48s.  Last read position: 18:48,921,880.  Last read name: A00175:147:H3MH3DMXY:2:1164:6605:22326
INFO    2021-11-26 12:12:34 SamFileValidator    Validated Read    70,000,000 records.  Elapsed time: 00:05:41s.  Time for last 10,000,000:   48s.  Last read position: 3:30,039,304.  Last read name: A00175:147:H3MH3DMXY:2:1307:23104:14920
INFO    2021-11-26 12:13:22 SamFileValidator    Validated Read    80,000,000 records.  Elapsed time: 00:06:30s.  Time for last 10,000,000:   48s.  Last read position: 16:21,419,589.  Last read name: A00175:147:H3MH3DMXY:2:1438:20717:23281
INFO    2021-11-26 12:14:11 SamFileValidator    Validated Read    90,000,000 records.  Elapsed time: 00:07:18s.  Time for last 10,000,000:   48s.  Last read position: 20:63,555,689.  Last read name: A00175:147:H3MH3DMXY:2:2181:19497:15468
INFO    2021-11-26 12:14:59 SamFileValidator    Validated Read   100,000,000 records.  Elapsed time: 00:08:07s.  Time for last 10,000,000:   48s.  Last read position: 3:187,229,812.  Last read name: A00175:147:H3MH3DMXY:2:2341:1994:27461
HISTOGRAM   java.lang.String
Error Type  Count
ERROR:INVALID_INDEX_FILE_POINTER    1
ERROR:MISMATCH_FLAG_MATE_NEG_STRAND 842976
[Fri Nov 26 12:15:47 UTC 2021] picard.sam.ValidateSamFile done. Elapsed time: 8.92 minutes.
Runtime.totalMemory()=2587885568
To get help, see http://broadinstitute.github.io/picard/index.html#GettingHelp`

[Pierre Lindenbaum]

INVALID_INDEX_FILE_POINTER

you should try to re-sort and/or re-index your file.

[Pierre Lindenbaum]

your bam is not sorted. you got Last read position: 16:2,971,202 and then Last read position: 19:50,278,169 and then chr16 again 16:31,473,305 ...