Note this release changes the default output CRAM version from 3.0 to 3.1. HTSlib and SAMtools have been able to read CRAM 3.1 since version 1.12, however other tools may not yet be able to cope. We know Noodles reads CRAM3.1 and htsjdk has a draft implementation, but not yet released.
HTSlib has options for modifying the output formats, which are exposed in SAMtools. When specifying an output format you can explicitly change the version via e.g. samtools view -O cram,version=3.0 ....
Further documentation on this change can be found at https://www.htslib.org/benchmarks/CRAM.html
HTSlib no longer fetches CRAM reference data from EBI's server by default. Your organisation may wish to set up local infrastructure to supply reference sequences, e.g., using the new ref-cache tool included in this HTSlib release. See the REF_CACHE and REF_PATH environment variables documented in https://www.htslib.org/doc/reference_seqs.html and the SAMtools manpage for details.
New work and changes:
New samtools checksum command. This checksums sequence, name, quality and barcode tags in an order and orientation agnostic manner, to facilitate validation of no data loss between raw fastq (or unmapped crams) through alignment, duplication marking, sorting, and other processing operations to get to the final aligned bam/cram. (PR #2122)
Extend samtools sort -M to distinguish between mapped and unmapped files. (PR #2110, fixes #2105. Reported by Armin Töpfer)
Allow the samtools sort "merging from..." message to be silenced. Setting the verbosity to 0 or 1 will now silence this message. (PR #2197, resolves #2185. Requested by Alex Predeus)
Add --save-counts option to samtools view. Adds an option to store counts of records processed, accepted and rejected by filtering to a file. (PR #2120, resolves #2038. Requested by Chang Y)
samtools fasta and fastq can now make faidx/fqidx indexes while writing using the --write-index option. (PR #2125, resolves #2118. Requested by Filipe G. Vieira)
Add a warning for samtools fastq on coordinate sorted data. (PR #2176, fixes #2169 and #2161. Reported by wook2014)
samtools tview add -i to hide inserts. (PR #2123. Thanks to Benjamin Bræstrup Sayoc)
Show optional headers with samtools bedcov -H. (PR #2140, fixes #2126. Reported by biounix)
samtools consensus now supports proper multi-threading. Previously this was restricted to decompression only, but it should now scale better. (PR #2174, supersedes PR #2141)
Add samtools consensus -T ref.fa functionality. This reports the reference value if a consensus value cannot be calculated. (PR #2153, fixes an additional request in #1915)
In samtools consensus, do not use consensus N for "*" (absent) calls that are masked due to insufficient depth. (PR #2204, fixes #2167. Reported by sanschiffre)
Improve plot-bamstats quality plots. (PR #2143 combined with PR #2116 (thanks to James Gilbert))
Make reheader -h use /tmp and honour TMPDIR. (PR #2168, related to #2165. Reported by Zhang Yuanfeng)
Set sort order header tag to unsorted when ordering is lost during samtools merge. (PR #2173, fixes #2159. Reported by Filipe G. Vieira)
Protect against merging CRAM files with different headers. (PR #2220, fixes #2218. Reported by Kevin Lewis)
samtools stats bug-fix to checksum calculation for quality values. This corrects the checksums but in turn makes the calculated value different to that reported by previous samtools versions. (PR #2193, fixes #2187)
Clarification for samtools stats when used on files with different sort orders. (PR #2198, fixes #2177. Reported by Filipe G. Vieira)
In samtools stats, dovetailed (completely overlapping) read pairs are now always counted as inward-oriented. Previously they could have been inwards or outwards depending on read ordering. (PR #2216, resolves #2210. Requested by Pontus Hüer)
Documentation:
Correct the example for 1:1 samtools consensus coords. (PR #2113, fixes #2111. Reported by schorlton-bugseq)
Documents the fastq format options used in SAMtools and HTSlib. (PR #2123, fixes #2121)
Remove mention of threads from samtools cat man page. (PR #2162, fixes #2160. Reported by Brandon Pickett)
Update samtools merge man page to include --template-coordinate. (PR #2164. Thanks to Nils Homer)
Revised CRAM reference sequence documentation in the samtools man page. (PR #2178)
Added fish shell completion and renamed completion for bash shell. These files can be copied to appropriate directories by the user. For full functionality it requires Python3.5+ and installed samtools manpages. (PR #2203. Thanks to LunarEclipse363)
Fix URL printed by the seq_cache_populate.pl script. (PR #2222. Thanks to Charles Plessy)
Bug fixes:
samtools consensus previously could give different results for BAM and CRAM files with the same content. This was because MD/NM tag generation was disabled in CRAM, but the decode_md=0 option did nothing with BAM. Note with --no-adj-MQ both BAM and CRAM gave identical results. Now use --input-fmt-option decode_md=0 to get the old CRAM behaviour. Otherwise, both BAM and CRAM will be utilising MD/NM to locally modify mapping quality. (PR #2156)
samtools consensus without -a previously still padded with leading Ns in some cases. It now consistently removes both leading and trailing Ns. Use "-a" if you want all reference bases displayed. (Part of PR #2174 above)
Change how markdup looks for single reads. Due to changes to fixmate in 1.21 markdup no longer recognised single reads that would have normally have been part of a pair. (PR #2117, fixes #2117. Reported by Kristy Horan)
Fix samtools merge crash on BAM files with malformed headers. (PR #2128, fixes #2127. Reported by Frostb1te)
Fix faidx --write-index invalid free. (PR #2147, fixes 2142. Reported by Alex Leonard)
Fix samtools fastq -i to force CRAM aux tag decoding. (PR #2155, fixes #2155. Reported by Alex Leonard)
Non user-visible changes and build improvements:
Improve htslib#PRnum support for Cirrus-CI and GitHub Actions. (PR #2115)
Fix broken tests due to MSYS2 changes. Due to changes in how MSYS2 perl reported the identity of the OS it was built for, our tests were failing to adapt to the Windows style file locations. (PR #2196)
Upgrade to _XOPEN_SOURCE=700, to match HTSlib. Also replace usleep() with nanosleep(). (PR #2221)
Download the source code here: samtools-1.22.tar.bz2.(The "Source code" downloads are generated by GitHub and are incomplete as they don't bundle HTSlib and are missing some generated files.)
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