Error encounterd querying alignments

[Yijun-Tian]

Hello IGV creator,

I want to load some single-cell RNA-seq BAM file to igv (linux 2.16.0, with carryon java), and after drag and release, the coverage, junctions and the alignment panel showed up correctly. But when I picked a specific gene, and click "go", a dialogue box appears says:
"Error encountered querying alignments: java.lang.StringIndexOutOfBoundsException: String index out of range: 1"
After click the OK, some alignments can show up, but I am not sure if the demonstration are error free.
I have switch the machine, but both centos and ubuntu showed the same error.

Below is the igv.log when error box jumps:
INFO [Mar 23,2023 13:27] [CommandListener] Listening on port 60151 INFO [Mar 23,2023 13:27] [GenomeManager] Loading genome: /home/user/igv/genomes/hg38.json INFO [Mar 23,2023 13:27] [TrackLoader] Loading resource: https://s3.amazonaws.com/igv.org.genomes/hg38/ncbiRefSeq.txt.gz INFO [Mar 23,2023 13:27] [TrackLoader] Loading resource: /mnt/raid0/Yijun_Tian/ATAC_GEX/multiome_GEX/22Rv1D.exonic.bam SEVERE [Mar 23,2023 13:27] [AlignmentTileLoader] Error loading alignment data SEVERE [Mar 23,2023 13:27] [AlignmentTileLoader] java.lang.StringIndexOutOfBoundsException: String index out of range: 1 at java.base/java.lang.StringLatin1.charAt(Unknown Source) at java.base/java.lang.String.charAt(Unknown Source) at org.igv/org.broad.igv.sam.mods.BaseModificationUtils.getBaseModificationSets(BaseModificationUtils.java:76) at org.igv/org.broad.igv.sam.SAMAlignment.getBaseModificationSets(SAMAlignment.java:338) at org.igv/org.broad.igv.sam.mods.BaseModificationCounts.incrementCounts(BaseModificationCounts.java:81) at org.igv/org.broad.igv.sam.BaseAlignmentCounts.incCounts(BaseAlignmentCounts.java:132) at org.igv/org.broad.igv.sam.AlignmentTileLoader$AlignmentTile.addRecord(AlignmentTileLoader.java:494) at org.igv/org.broad.igv.sam.AlignmentTileLoader.loadTile(AlignmentTileLoader.java:245) at org.igv/org.broad.igv.sam.AlignmentDataManager.loadInterval(AlignmentDataManager.java:391) at org.igv/org.broad.igv.sam.AlignmentDataManager.load(AlignmentDataManager.java:338) at org.igv/org.broad.igv.sam.CoverageTrack.load(CoverageTrack.java:186) at org.igv/org.broad.igv.ui.IGV.lambda$repaint$16(IGV.java:2267) at java.base/java.util.concurrent.CompletableFuture$AsyncRun.run(Unknown Source) at java.base/java.util.concurrent.ThreadPoolExecutor.runWorker(Unknown Source) at java.base/java.util.concurrent.ThreadPoolExecutor$Worker.run(Unknown Source) at java.base/java.lang.Thread.run(Unknown Source)

I also attached a bam file to reproduce this error, you may try jump to "chr1:165,905-165,944" to meet the error.
error.zip

Thanks,
Yijun

[jrobinso]

The error is caused by the presence of an "MM" tag in the alignment records. "MM" is a reserved tag for base modifications (see section 1.7 here): https://samtools.github.io/hts-specs/SAMtags.pdf. The use in this file is for something else, it does not conform to the expected pattern. Do you know what "MM" is used for here, and also what software produced this file?

[Yijun-Tian]

Thanks for the sharp comment! The BAM is generated from cellranger-arc, and they do have a MM tag listed in this documentation:
https://support.10xgenomics.com/single-cell-multiome-atac-gex/software/pipelines/latest/output/bam-gex

Could IGV recognize the tag type, too, before parsing the base modification info? The 10x MM tag type is an integer and the base modification is a string. What a coincidence.

[jrobinso]

Thanks for bringing that to my attention. I don't think this is a legal use for MM, and 10X should fix it, but since these files are being produced I will see what I can do to support it.

@jkbonfield Thoughts on this 10X use of "MM"? My understanding was all cap tags are reserved. Its a conundrum if MM can mean anything. In this specific case I can recognize it by type.

https://support.10xgenomics.com/single-cell-multiome-atac-gex/software/pipelines/latest/output/bam-gex

[jkbonfield]

I can confirm this tag breaks the SAM specification. There are many more choices of private user-space tags than there are reserved tags, so it's disappointing when people use the reserved tags for their own purposes.

This is something to raise with the 10x genomics support. They need to fix their software.

[Yijun-Tian]

I just tested the 2.9.5 version is OK with the 10x BAM file. Seems this is the newest version not aware of the MM tag. From: James Bonfield ***@***.***> Sent: Friday, March 24, 2023 4:29 AM To: igvteam/igv ***@***.***> Cc: Tian, Yijun ***@***.***>; State change ***@***.***> Subject: EXT:Re: [igvteam/igv] Error encounterd querying alignments (Issue #1305) I can confirm this tag breaks the SAM specification. There are many more choices of private user-space tags than there are reserved tags, so it's disappointing when people use the reserved tags for their own purposes. This is something to raise with the 10x genomics support. They need to fix their software. — Reply to this email directly, view it on GitHub<#1305 (comment)>, or unsubscribe<https://github.com/notifications/unsubscribe-auth/ANRYRST6LXUNHA4QEVMBLETW5VLLXANCNFSM6AAAAAAWFQ2YCY>. You are receiving this because you modified the open/close state.Message ID: ***@***.******@***.***>> This transmission may be confidential or protected from disclosure and is only for review and use by the intended recipient. Access by anyone else is unauthorized. Any unauthorized reader is hereby notified that any review, use, dissemination, disclosure or copying of this information, or any act or omission taken in reliance on it, is prohibited and may be unlawful. If you received this transmission in error, please notify the sender immediately. Thank you.