Cannot convert CRAM back to BAM

[loganjeverett]

I am getting an error when I try to convert any CRAM file back to BAM format. I am using the exact same version of cramtools (3.0) and same genome reference fasta file to do BAM -> CRAM conversion and CRAM -> BAM conversion. I created a test file to confirm that the problem is reproducible.

To convert BAM to CRAM, I run:
java -jar ~/Tools/cramtools/cramtools-3.0.jar cram -I test.bam -R /data/projectdata/htt/genomes/hg38/hg38.fa -O test.cram
That works without issue, but then I run CRAM back to BAM and get:
java -jar ~/Tools/cramtools/cramtools-3.0.jar bam -I test.cram -R /data/projectdata/htt/genomes/hg38/hg38.fa -O test_uncram.bam Exception in thread "main" java.lang.reflect.InvocationTargetException at sun.reflect.NativeMethodAccessorImpl.invoke0(Native Method) at sun.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:62) at sun.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43) at java.lang.reflect.Method.invoke(Method.java:498) at net.sf.cram.CramTools.invoke(CramTools.java:91) at net.sf.cram.CramTools.main(CramTools.java:121) Caused by: java.lang.RuntimeException: java.io.FileNotFoundException: http://www.ebi.ac.uk/ena/cram/md5/2648ae1bacce4ec4b6cf337dcae37816 at net.sf.cram.ref.ReferenceSource.getRegion(ReferenceSource.java:198) at net.sf.cram.Cram2Bam.main(Cram2Bam.java:218) ... 6 more Caused by: java.io.FileNotFoundException: http://www.ebi.ac.uk/ena/cram/md5/2648ae1bacce4ec4b6cf337dcae37816 at sun.net.www.protocol.http.HttpURLConnection.getInputStream0(HttpURLConnection.java:1872) at sun.net.www.protocol.http.HttpURLConnection.getInputStream(HttpURLConnection.java:1474) at java.net.URL.openStream(URL.java:1045) at net.sf.cram.ref.ReferenceSource.loadFromPath(ReferenceSource.java:317) at net.sf.cram.ref.ReferenceSource.findBasesByMD5(ReferenceSource.java:363) at net.sf.cram.ref.ReferenceSource.getRegion(ReferenceSource.java:194) ... 7 more
As best I can tell, it's checking the ENA CRAM reference registry, even though I provided a local copy of the reference file. The --skip-md5-check option doesn't seem to help, just gets to this error faster. I also tried cloning and recompiling the latest version (3.0-b203) from github, that did not help either.

[vadimzalunin]

Is the reference indexed?

…

On Wed, 27 Sep 2017, 22:12 loganjeverett ***@***.***> wrote: I am getting an error when I try to convert any CRAM file back to BAM format. I am using the exact same version of cramtools (3.0) and same genome reference fasta file to do BAM -> CRAM conversion and CRAM -> BAM conversion. I created a test file to confirm that the problem is reproducible. To convert BAM to CRAM, I run: java -jar ~/Tools/cramtools/cramtools-3.0.jar cram -I test.bam -R /data/projectdata/htt/genomes/hg38/hg38.fa -O test.cram That works without issue, but then I run CRAM back to BAM and get: java -jar ~/Tools/cramtools/cramtools-3.0.jar bam -I test.cram -R /data/projectdata/htt/genomes/hg38/hg38.fa -O test_uncram.bam Exception in thread "main" java.lang.reflect.InvocationTargetException at sun.reflect.NativeMethodAccessorImpl.invoke0(Native Method) at sun.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:62) at sun.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43) at java.lang.reflect.Method.invoke(Method.java:498) at net.sf.cram.CramTools.invoke(CramTools.java:91) at net.sf.cram.CramTools.main(CramTools.java:121) Caused by: java.lang.RuntimeException: java.io.FileNotFoundException: http://www.ebi.ac.uk/ena/cram/md5/2648ae1bacce4ec4b6cf337dcae37816 at net.sf.cram.ref.ReferenceSource.getRegion(ReferenceSource.java:198) at net.sf.cram.Cram2Bam.main(Cram2Bam.java:218) ... 6 more Caused by: java.io.FileNotFoundException: http://www.ebi.ac.uk/ena/cram/md5/2648ae1bacce4ec4b6cf337dcae37816 at sun.net.www.protocol.http.HttpURLConnection.getInputStream0(HttpURLConnection.java:1872) at sun.net.www.protocol.http.HttpURLConnection.getInputStream(HttpURLConnection.java:1474) at java.net.URL.openStream(URL.java:1045) at net.sf.cram.ref.ReferenceSource.loadFromPath(ReferenceSource.java:317) at net.sf.cram.ref.ReferenceSource.findBasesByMD5(ReferenceSource.java:363) at net.sf.cram.ref.ReferenceSource.getRegion(ReferenceSource.java:194) ... 7 more As best I can tell, it's checking the ENA CRAM reference registry, even though I provided a local copy of the reference file. The --skip-md5-check option doesn't seem to help, just gets to this error faster. I also tried cloning and recompiling the latest version (3.0-b203) from github, that did not help either. — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#73>, or mute the thread <https://github.com/notifications/unsubscribe-auth/AAoz5IyLUAyGbMwnJ7kY-PmV8hC30Y8Tks5smroygaJpZM4PmZ49> .

[loganjeverett]

Yes, there is a corresponding .fai file (hg38.fa.fai) created with samtools faidx. I have also now tried cramtools-2.1.jar and that converts in both directions without an error. It is possible that the cramtools 3.0 is inserting some incorrect URIs into the header, even though I did NOT specify the --inject-sq-uri option. Is there a way to view the cram file header without doing the full conversion back to bam?

[vadimzalunin]

please try the -H option