I have not been able to get GATK 3.7 HaplotypeCaller to work with CRAM files at all (it has a 100% failure rate so far with our whole genome CRAMs). Based on my analysis of the problem, I don't think GATK 3.7 will work with any CRAM files containing IUPAC ambiguity codes other than 'N' (including GRCh37/hs37d5 and GRCh38/HS38DH).
The error I get is:
ERROR 2017-01-05 02:18:59 Slice Reference MD5 mismatch for slice 2:60825966-60861215, ATCTTTCATG...CTCTCCCATT
##### ERROR ------------------------------------------------------------------------------------------
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##### ERROR
##### ERROR MESSAGE: SAM/BAM/CRAM file /keep/46909b690725869e1d9bfbc1da4a1398+19932/20657_7.cram is malformed. Please see https://software.broadinstitute.org/gatk/documentation/article?id=1317for more
##### ERROR ------------------------------------------------------------------------------------------
This error occurs for 100% of my CRAM files, which can be read by samtools, scramble, or previous versions of GATK (including 3.6) without any issues, so the error message is incorrect and the CRAM files are not malformed.
The CRAM slice in question is on chromosome 3 of hs37d5 (3:60825966-60861215). We can verify externally that the FASTA reference we are passing into GATK with -R does have the md5 that GATK reports it is expecting:
$ samtools faidx /keep/d527a0b11143ebf18be6c52ff6c09552+2163/hs37d5.fa 3:60825966-60861215 | grep -v '^>' | tr -d '\012' | md5sum
0e0ff678755616cba9ac362f15b851cc -
And the sequence starts and ends with the bases that htsjdk reports:
$ samtools faidx /keep/d527a0b11143ebf18be6c52ff6c09552+2163/hs37d5.fa 3:60825966-60861215 | grep -v '^>' | tr -d '\012' | cut -c1-10
ATCTTTCATG
$ samtools faidx /keep/d527a0b11143ebf18be6c52ff6c09552+2163/hs37d5.fa 3:60825966-60861215 | grep -v '^>' | tr -d '\012' | cut -c35241-
CTCTCCCATT
I ended up having to recompile GATK and htsjdk from source and added some print debugging to htsjdk to dump the whole sequence from which the md5 was being calculated. It seems the sequence that cause problems are regions of the reference with IUPAC ambiguity codes other than 'N' (in this case a slice of chromosome 3 that contains an 'M' and two 'R's). In GATK 3.7 (built with htsjdk 2.8.1), the reference which is used to calculate the md5 for the slice has had all ambiguity codes converted to 'N'. The md5 it calculates for this slice (according to my print debugging) is: 5d820b3624e78202f503796f7330d8d9
I have verified that this is the md5 we would get from converting the IUPAC codes in this slice to N's:
$ samtools faidx /keep/d527a0b11143ebf18be6c52ff6c09552+2163/hs37d5.fa 3:60825966-60861215 | grep -v '^>' | tr -d '\012' | tr RYMKWSBDHV NNNNNNNNNN | md5sum
5d820b3624e78202f503796f7330d8d9 -
I have tried in vain to figure out where in GATK and/or htsjdk the ambiguous reference bases are being converted to 'N's. I initially thought that it was in the CachingIndexedFastaSequenceFile call to BaseUtils.convertIUPACtoN (when preserveIUPAC is false, although I didn't find any code path that could set it to true). However, after recompiling with preserveIUPAC manually set to true, the problem persisted. I guess there must be some other place where the bases are remapped. I'll leave it to you guys to figure out how to get an unmodified view on the reference for htsjdk to use for CRAM decoding.
There is, however, no mystery as to why this problem has suddenly appeared in GATK 3.7. The slice md5 validation code in htsjdk was only added in July 2016 (https://github.com/samtools/htsjdk/commit/a781afa9597dcdbcde0020bfe464abee269b3b2e). The first release version it appears in is version 2.7.0. Prior to that, it seems CRAM slice md5's were not validated in htsjdk, so this error would not have occurred.