ValueError: More than one match to b'ASC.' / b'XSC.'

[moldovannorbert]

When running xenomapper2 on bams mapped by bwa mem I get the following errors from pylazybam:

xenomapper2 v2.0rc1 --primary=/scratch-shared/fmlab/nmoldovan/tmp/FM_seq_020/trimmed/7_xenomapper/1_mapping/LP0051_08_L001_primary.bam --secondary=/scratch-shared/fmlab/nmoldovan/tmp/FM_seq_020/trimmed/7_xenoma$

Traceback (most recent call last):
File "/gpfs/home1/norbertm/projects/code/core_pipeline_310322/workflow/rules/7_xenomapper/.snakemake/conda/39b62994/bin/xenomapper2", line 8, in
sys.exit(main())
File "/gpfs/home1/norbertm/projects/code/core_pipeline_310322/workflow/rules/7_xenomapper/.snakemake/conda/39b62994/lib/python3.10/site-packages/xenomapper2/cli.py", line 193, in main
pair_counts, counts, writer = xenomap(primary_bam,
File "/gpfs/home1/norbertm/projects/code/core_pipeline_310322/workflow/rules/7_xenomapper/.snakemake/conda/39b62994/lib/python3.10/site-packages/xenomapper2/xenomapper2.py", line 884, in xenomap
forward_state, reverse_state = xenomap_states(primary_aligns,
File "/gpfs/home1/norbertm/projects/code/core_pipeline_310322/workflow/rules/7_xenomapper/.snakemake/conda/39b62994/lib/python3.10/site-packages/xenomapper2/xenomapper2.py", line 685, in xenomap_states
prim_f_AS, prim_f_XS = score_function(prim_f_aligns,
File "/gpfs/home1/norbertm/projects/code/core_pipeline_310322/workflow/rules/7_xenomapper/.snakemake/conda/39b62994/lib/python3.10/site-packages/xenomapper2/xenomapper2.py", line 314, in get_bamprimary_AS_XS
AS = AS_function(bamprimary[0])
File "/gpfs/home1/norbertm/projects/code/core_pipeline_310322/workflow/rules/7_xenomapper/.snakemake/conda/39b62994/lib/python3.10/site-packages/pylazybam/tags.py", line 55, in get_AS
raise ValueError(
ValueError: More than one match to b'ASC.' was found in b'I\x01\x00\x00\x04\x00\x00\x00ASC\x01'<V\x17\x01\x00S\x00\x97\x00\x00\x00\x04\x00\x00\x00!SC\x01I\xff\xff\xffA01685:16:HGHWVDSX3:1:1258:25373:19100\x00p$

or

xenomapper2 v2.0rc1 --primary=/scratch-shared/fmlab/nmoldovan/tmp/FM_seq_020/trimmed/7_xenomapper/1_mapping/LP0051_07_L001_primary.bam --secondary=/scratch-shared/fmlab/nmoldovan/tmp/FM_seq_020/trimmed/7_xenoma$

Traceback (most recent call last):
File "/gpfs/home1/norbertm/projects/code/core_pipeline_310322/workflow/rules/7_xenomapper/.snakemake/conda/39b62994/bin/xenomapper2", line 8, in
sys.exit(main())
File "/gpfs/home1/norbertm/projects/code/core_pipeline_310322/workflow/rules/7_xenomapper/.snakemake/conda/39b62994/lib/python3.10/site-packages/xenomapper2/cli.py", line 193, in main
pair_counts, counts, writer = xenomap(primary_bam,
File "/gpfs/home1/norbertm/projects/code/core_pipeline_310322/workflow/rules/7_xenomapper/.snakemake/conda/39b62994/lib/python3.10/site-packages/xenomapper2/xenomapper2.py", line 884, in xenomap
forward_state, reverse_state = xenomap_states(primary_aligns,
File "/gpfs/home1/norbertm/projects/code/core_pipeline_310322/workflow/rules/7_xenomapper/.snakemake/conda/39b62994/lib/python3.10/site-packages/xenomapper2/xenomapper2.py", line 685, in xenomap_states
prim_f_AS, prim_f_XS = score_function(prim_f_aligns,
File "/gpfs/home1/norbertm/projects/code/core_pipeline_310322/workflow/rules/7_xenomapper/.snakemake/conda/39b62994/lib/python3.10/site-packages/xenomapper2/xenomapper2.py", line 315, in get_bamprimary_AS_XS
XS = XS_function(bamprimary[0])
File "/gpfs/home1/norbertm/projects/code/core_pipeline_310322/workflow/rules/7_xenomapper/.snakemake/conda/39b62994/lib/python3.10/site-packages/pylazybam/tags.py", line 108, in get_XS
raise ValueError(
ValueError: More than one match to b'XSC.' was found in b'N\x01\x00\x00\x07\x00\x00\x00XSC\x02'<V\x1b\x01\x00S\x00\x97\x00\x00\x00\x07\x00\x00\x00QSC\x02b\xff\xff\xffA01685:16:HGHWVDSX3:1:1161:21965:27946\x00p$

I am mapping and sorting reads in the previous step by:

bwa mem {params.ref} {input}
-M
-t {threads}
2> {log} |
samtools sort
-n
-@ {threads}
-o {output} 2>> {log}

Intriguingly this pipeline worked with another batch of files already, but now it is throwing the above error.

Edit: I forgot to add, that we are talking about 150 bp PE seq of genomic data.

[genomematt]

I have had a quick look and can't see the obvious problem of there being some other information in the string that gets picked up by the regex search.
If you use samtool view are these the first reads in the file?
The easiest way to proceed if you don't mind is to send me the files so I can reproduce the error. If you can use samtools view input.bam "chrom:start-end" > output.bam with some appropriate region to make a small file that reproduces that would be even better.
If you can't do that, could send me the full SAM for that specific read. You can extract this with samtools view file.bam | grep -m1 "A01685:16:HGHWVDSX3:1:1161:21965:27946" -

[ivanbelzen]

Hi! I'm encountering the same error

Error message:
ValueError: More than one match to b'XSC.' was found in b'_\x01\x00\x00\x00\x00\x00\x00XSC\x07(,V/\x01\x00S\x00\x96\x00\x00\x00\x00\x00\x00\x00\xe4RC\x07\xf6\xfe\xff\xffLH00659:469:2357J3LT4:4:1124:26281:5190\x00`\t\x00\x00A\x81\x18\x88\x18\x81\x88AA\x18\x84\x88\x88D(\x12"\x81D\x88\x88\x84\x88\x88\x12!\x81!\x11\x88AA\x18\x81\x88(\x88\x11H\x18(HA\x14\x11\x18H\x88\x81\x11\x11(\x88\x18DD\x18\x84\x81\x88A\x18(HD\x18\x82\x11\x84\x11(\x88D\x81\x14((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((ASS$\x01XSCCXNC\x00XMC\x01XOC\x00XGC\x00NMC\x01MDZ31T118\x00YSS&\x01YTZCP\x00'meaning that more than one value of the tag is presentor that another tag contains a match as part of its value

Reads from sam file:

LH00659:469:2357J3LT4:4:1124:26281:5190 163 chr1 121852645 44 147M3S = 121852761 266 CTCTCCTTATTGTTGGGCATTTTGGTTAAGGTCACCCCCATTGATTCTTGAGAGTTTCTCACCTCCCACGTCTCTGGAACTTTCTAGAGGATCCCCCCAACCTCACATCCCCAGAAGATAATTTATTATTGAGAATTGTTTTGGCTACCC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:294 XS:i:78 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:147 YS:i:292 YT:Z:CP
LH00659:469:2357J3LT4:4:1124:26281:5190 83 chr1 121852761 44 150M = 121852645 -266 GATAATTTATTATTGAGAATTGTTTTGGCTACCCTAGGTTTTTGTTTTACCATACAAATTGAGAATTATTCTTTAAGTATCTGTGAAGAAATGTTTTAAAAACTTTATGGGGATTGTATTGAATCTGTGGATTCAATGAACTTTGGTAAG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:292 XS:i:67 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:31T118 YS:i:294 YT:Z:CP

The data has been mapped with bowtie to hg38 and to mm39 as specified in the worked example on this github

bowtie2 --local --threads 7 --reorder
-x genomes/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.bowtie_index
-1 Tumor_C10_1.fq.gz
-2 Tumor_C10_2.fq.gz > bams/Tumor_C10.hg38.bowtie.sam

[ivanbelzen]

another occurrance of this error

LH00659:469:2357J3LT4:4:2144:48464:13485 99 chrX 155407193 39 150M = 155407388 345 TGTTTTTGTGCCAGTACCATGCTGTTTTGGTTACTGTAGCCTTGTAGTATAGTTTCAGGTCAGGTAGTGTGATGCCTCCAGCTTTGTTCTTTTTGCTTAGGGTTACTTTGGTGATTCATGCTCTTTTTTGGTTGCATATGAATTTTAGAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:300 XS:i:112 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:150 YS:i:300 YT:Z:CP
LH00659:469:2357J3LT4:4:2144:48464:13485 147 chrX 155407388 39 150M = 155407193 -345 GGAATAGCATTGAATCTATAAATTTCTTTGGTCAGTATGGCCATTTTAACAATATTAATTATTTCAATTCATAAGCATGGGGTGTTTTACCATTATGTTTGTGTCATCTCTGATTTATCTCAGCAGTGTTTTATAGTTCTCCTTGTAGAG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:300 XS:i:95 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:150 YS:i:300 YT:Z:CP

[genomematt]

Bowtie now appends the pair tag to the bam so there are two XC tags in the bam. As a temporary hack you should be able to covert to SAM and back to BAM and it should remove. I will be able to do a more permanent fix early in June.

…

--=-=--=-=--=-=--=-=--=-=--=-=--=-=--=-=--=-=--=-=--=-=--=-=--=-=--=-=--=-=--=-= A/Prof Matthew Wakefield PhD Stafford Fox Principal Research Fellow of Ovarian Cancer Biology and Genomics Bioinformatics, WEHI ***@***.*** The Royal Women’s Hospital and Mercy Hospital for Women Department of Obstetrics, Gynaecology, and Newborn Health. University of Melbourne ***@***.*** M +61 402 916 018

________________________________ From: ivanbelzen ***@***.***> Sent: Friday, May 15, 2026 9:55:36 PM To: genomematt/xenomapper2 ***@***.***> Cc: Matthew Wakefield ***@***.***>; Assign ***@***.***> Subject: Re: [genomematt/xenomapper2] ValueError: More than one match to b'ASC.' / b'XSC.' (Issue #11) [https://avatars.githubusercontent.com/u/50848424?s=20&v=4]ivanbelzen left a comment (genomematt/xenomapper2#11)<https://urldefense.com/v3/__https://github.com/genomematt/xenomapper2/issues/11*issuecomment-4459524022__;Iw!!KKt0acMt!cBnH9Vemjl14RNZK6jd1jCXm7mCuRz6268aFyMMLN5SgmITW9VWkBVNIDF8_2eXGASv1OBAira9BkG4_gNj6J9Qcm7NO$> another occurrance of this error LH00659:469:2357J3LT4:4:2144:48464:13485 99 chrX 155407193 39 150M = 155407388 345 TGTTTTTGTGCCAGTACCATGCTGTTTTGGTTACTGTAGCCTTGTAGTATAGTTTCAGGTCAGGTAGTGTGATGCCTCCAGCTTTGTTCTTTTTGCTTAGGGTTACTTTGGTGATTCATGCTCTTTTTTGGTTGCATATGAATTTTAGAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:300 XS:i:112 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:150 YS:i:300 YT:Z:CP LH00659:469:2357J3LT4:4:2144:48464:13485 147 chrX 155407388 39 150M = 155407193 -345 GGAATAGCATTGAATCTATAAATTTCTTTGGTCAGTATGGCCATTTTAACAATATTAATTATTTCAATTCATAAGCATGGGGTGTTTTACCATTATGTTTGTGTCATCTCTGATTTATCTCAGCAGTGTTTTATAGTTCTCCTTGTAGAG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:300 XS:i:95 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:150 YS:i:300 YT:Z:CP — Reply to this email directly, view it on GitHub<https://urldefense.com/v3/__https://github.com/genomematt/xenomapper2/issues/11*issuecomment-4459524022__;Iw!!KKt0acMt!cBnH9Vemjl14RNZK6jd1jCXm7mCuRz6268aFyMMLN5SgmITW9VWkBVNIDF8_2eXGASv1OBAira9BkG4_gNj6J9Qcm7NO$>, or unsubscribe<https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/AARWWQQRTIBQXLH4DL7PECL424ATRAVCNFSM6AAAAACY7HLH3CVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHM2DINJZGUZDIMBSGI__;!!KKt0acMt!cBnH9Vemjl14RNZK6jd1jCXm7mCuRz6268aFyMMLN5SgmITW9VWkBVNIDF8_2eXGASv1OBAira9BkG4_gNj6J7tuYDYb$>. You are receiving this because you were assigned.Message ID: ***@***.***>

[ivanbelzen]

Thank you for the input! I have tried this (see below) but unfortunately it still gives the same error

samtools view -h bowtie.bam -@ 4 | samtools view -@4 -b -o bowtie.redo.bam