Loading CRAM files failed with release 2.16.0

[antonylebechec]

Dear IGVTeam,

Dealing with CRAM files, I used to open them with IGV (release 2.12.13). However, IGV release 2.16.0 doesn't open correctly CRAM files.

These CRAM files were generated using samtools (1.15.1) and these parameters: "version=3.0,level=9,no_ref" (version 3.0, compression level 9 and include references, i.e. all bases, no need reference genome)
and are indexed in crai

OS: Mac (intel)
IGV: MacApp 2.16.0 (with Java)

I tried also releases 2.13.2, 2.14.0, 2.15.4, and they failed too.

Do you have more information about this issue?

IGV [2.16.0]

IGV [2.12.13]

[jrobinso]

It's hard to say what the problem is with screenshots, and this is the first report of this we have had. All crams that I have tried load without error. Could you share your igv.log file, it should have some diagnostic information. Its located in an "igv" folder under your home directory, and will have a name like. "igv0.log" or similiar. There could be several, we just need the log for the time you experienced the issue. You can zip and attach here, or if you'd rather not have it public email to igvteam (at) broadinstitute.org

[antonylebechec]

Dear @jrobinso,

Sure, screenshots were just to illustrate, in case you already had this issue.

So, here is the log file (IGV 2.16.0):

INFO [févr. 17,2023 12:20] [Main] Startup  IGV Version 2.16.0 01/25/2023 01:50 PM
INFO [févr. 17,2023 12:20] [Main] Java 11.0.13 (build 11.0.13+8) 2021-10-19
INFO [févr. 17,2023 12:20] [Main] Java Vendor: Eclipse Adoptium https://adoptium.net/
INFO [févr. 17,2023 12:20] [Main] JVM: OpenJDK 64-Bit Server VM Temurin-11.0.13+8
INFO [févr. 17,2023 12:20] [Main] OS: Mac OS X 13.1 x86_64
INFO [févr. 17,2023 12:20] [Main] IGV Directory: /Users/lebechea/igv
INFO [févr. 17,2023 12:20] [CommandListener] Listening on port 60151
INFO [févr. 17,2023 12:20] [GenomeManager] Loading genome: https://s3.amazonaws.com/igv.org.genomes/hg19/hg19.json
INFO [févr. 17,2023 12:20] [TrackLoader] Loading resource:  https://s3.amazonaws.com/igv.org.genomes/hg19/ncbiRefSeq.sorted.txt.gz
INFO [févr. 17,2023 12:21] [TrackLoader] Loading resource:  /Users/lebechea/STARK/output/repository/SOMATIC/SOLIDTUMOR/RUN_TEST_920/Sample4/STARK/Sample4.archive.cram
SEVERE [févr. 17,2023 12:21] [TrackLoader] null
SEVERE [févr. 17,2023 12:21] [TrackLoader] java.lang.NullPointerException
        at htsjdk@3.0.0/htsjdk.samtools.cram.structure.CRAMRecordReadFeatures.getByteOrDefault(CRAMRecordReadFeatures.java:498)
        at htsjdk@3.0.0/htsjdk.samtools.cram.structure.CRAMRecordReadFeatures.restoreReadBases(CRAMRecordReadFeatures.java:419)
        at htsjdk@3.0.0/htsjdk.samtools.cram.structure.CRAMCompressionRecord.restoreReadBases(CRAMCompressionRecord.java:468)
        at htsjdk@3.0.0/htsjdk.samtools.cram.structure.Slice.normalizeCRAMRecords(Slice.java:507)
        at htsjdk@3.0.0/htsjdk.samtools.cram.structure.Container.getSAMRecords(Container.java:322)
        at htsjdk@3.0.0/htsjdk.samtools.CRAMIterator.nextContainer(CRAMIterator.java:112)
        at htsjdk@3.0.0/htsjdk.samtools.CRAMIterator.hasNext(CRAMIterator.java:204)
        at htsjdk@3.0.0/htsjdk.samtools.SamReader$AssertingIterator.hasNext(SamReader.java:608)
        at org.igv/org.broad.igv.sam.reader.WrappedIterator.hasNext(WrappedIterator.java:49)
        at org.igv/org.broad.igv.sam.AlignmentUtils.firstAlignments(AlignmentUtils.java:243)
        at org.igv/org.broad.igv.sam.AlignmentDataManager.inferType(AlignmentDataManager.java:400)
        at org.igv/org.broad.igv.sam.AlignmentTrack.init(AlignmentTrack.java:352)
        at org.igv/org.broad.igv.track.TrackLoader.loadAlignmentsTrack(TrackLoader.java:966)
        at org.igv/org.broad.igv.track.TrackLoader.load(TrackLoader.java:161)
        at org.igv/org.broad.igv.ui.IGV.load(IGV.java:1249)
        at org.igv/org.broad.igv.ui.IGV.loadResources(IGV.java:1179)
        at org.igv/org.broad.igv.ui.IGV$2.run(IGV.java:409)
        at org.igv/org.broad.igv.util.LongRunningTask.call(LongRunningTask.java:72)
        at java.base/java.util.concurrent.FutureTask.run(Unknown Source)
        at java.base/java.util.concurrent.ThreadPoolExecutor.runWorker(Unknown Source)
        at java.base/java.util.concurrent.ThreadPoolExecutor$Worker.run(Unknown Source)
        at java.base/java.lang.Thread.run(Unknown Source)

SEVERE [févr. 17,2023 12:21] [IGV] Error loading track
SEVERE [févr. 17,2023 12:21] [IGV] java.lang.NullPointerException
        at org.igv/org.broad.igv.exceptions.DataLoadException.<init>(DataLoadException.java:38)
        at org.igv/org.broad.igv.track.TrackLoader.load(TrackLoader.java:272)
        at org.igv/org.broad.igv.ui.IGV.load(IGV.java:1249)
        at org.igv/org.broad.igv.ui.IGV.loadResources(IGV.java:1179)
        at org.igv/org.broad.igv.ui.IGV$2.run(IGV.java:409)
        at org.igv/org.broad.igv.util.LongRunningTask.call(LongRunningTask.java:72)
        at java.base/java.util.concurrent.FutureTask.run(Unknown Source)
        at java.base/java.util.concurrent.ThreadPoolExecutor.runWorker(Unknown Source)
        at java.base/java.util.concurrent.ThreadPoolExecutor$Worker.run(Unknown Source)
        at java.base/java.lang.Thread.run(Unknown Source)

Here are also the CRAM and CRAI files I used (just a test sample for devel):
Sample4.archive.cram.zip
Sample4.archive.cram.crai.zip

Thanks for your help!

[antonylebechec]

I just test release 2.16.0 on a Windows system. It seems that the same issue append.

igv0.log

[antonylebechec]

Another test with Samtools shoxs that the CRAM file can be view:

$ samtools view Sample4.archive.cram | head -n2
M01656:13:000000000-D02HD:1:1101:23237:26068	163	chr1	111984608	60	8S212M10S	=	111984608	212	CCGTATCTCCCTTCCCTGATTCCCAAAGGATTCCACTTCACACTTACAACTCAGAAAGGCTTGAGTCCAGCACAGCAAGTGAGCAGTCTTCACTGAGAGAGGCCAGGAAGGGAACACTATCAGATGGAGGAGAGGGTTGTGAAAGGTCCAGCCTGGATCCCAAGACAAAACCTTAACTGGGCCGGCCCTGCCGCCACTGTAAAGTGACCCAGGAGAGGCACGTCAGTGTG	<<;;;=?>?<<>>?==?@>=>?==@@@@???@?=<??@???@??=;?@>><>;?><2<>??@@;?>?=@@???@@=?=?>?>@=@?>??@?@??B?@?@>?>==@@??@@>>?@?@??<=?@?>?B??@>;?>;0;;=<>@?@@??<?<@@?=>B????<=@@@;>@@@@?<?@<@??B??==:>===?B<=:==<?<B><>>?>@>?==@?>>?>?>=@?:>?2;<?<2	BC:Z:CCGTAGTT-ACTTGAGT	MC:Z:8S212M10S	MD:Z:212	NM:i:0	MQ:i:60	OQ:Z:BBAAAFFFFFFFGGGGGGGGGGHHHHHHHHHHHH6GHHHFHHHHFBGGGFCGEGFB3BFHHHGEGHHHHHHFHGHGFFFGFFHGGFGFHHGHHHHHHHHGGGGGHHHHHHGGHHGHHHHGHHGGHHHHHGEFGE/E<FDGGHHHFHDGFHHHGFHHHHHGHGHHEHHHHHHGHHHHHHHHHGHGGGGGHHGHGGG@GBHGGGGGGGGGGGGGGGGFGGFGGGFG.ABFB/	AS:i:212	XS:i:21	RG:Z:1-371D323C
M01656:13:000000000-D02HD:1:1101:23237:26068	83	chr1	111984608	60	8S212M10S	=	111984608	-212	CCGTATCTCCCTTCCCTGATTCCCAAAGGATTCCACTTCACACTTACAACTCAGAAAGGCTTGAGTCCAGCACAGCAAGTGAGCAGTCTTCACTGAGAGAGGCCAGGAAGGGAACACTATCAGATGGAGGAGAGGGTTGTGAAAGGTCCAGCCTGGATCCCAAGAAAAAACCTTAACTGGGCCGGCCCTGCCGCCACTGTAAAGTGACCCAGGAGAGGCACGTCAGTGTG	>;>==<;2>>?>;>>???=<;>>2>>?=?=>>>@;;>>?>B>@@<<=@>?<???@@?=?@@@?????????=?>=<>??<?>=???????B>@@;???>?==???=<@?=<?@>B>@<??B????=??=???>==;@??@?@@?=?>>????@@=?=:>><@<?;0>@=<??==>>?0==;;9;;>>??;;:=?B>@@?<@@??@?>>>@?=????===<;>>?><<<<<	BC:Z:CCGTAGTT-ACTTGAGT	MC:Z:8S212M10S	MD:Z:157C54	NM:i:1	MQ:i:60	OQ:Z:FFFFGDE.GGGGEGGGFFGFAGF0GGGGGFGFGG;;GGFGHHHHGCCHGGCFHHHHGGHHHGHHHHHFGHFHFFGDGHGDFFGFHHHFHHHHHHBHHHGGHGHFGHCHGHHHHHHHHHHHHHHHFHGGHHHHFGGEHGHHFHHHHHGGFHHHHHHFF?GGDHDF>1GHF@HFFFGFF1GGEEEEAGGFFEEGGHHGHHHHHHHHHHHGGGGGGGGGGGCCCFFFFCCCCC	AS:i:207	XS:i:21	RG:Z:1-371D323C

Do you think there is a problem with the format? With tags on this file?

When I create a BAM file from the CRAM file, it can be loaded in IGV:

$ samtools view -h Sample4.archive.cram -o Sample4.archive.bam
$ samtools index Sample4.archive.bam

[jrobinso]

Thanks for the sample data, we will look into it.

[lbergelson]

@antonylebechec Thank you so much for the helpful bug report and the test data.

It's a simple bug in the underlying htsjdk library. I just built and tested a patch that should fix it. I'll have to release a new htsjdk and then incorporate that into IGV.

[antonylebechec]

Thanks a lot IGV Team!!!