CRAM 3.1: advances in the CRAM file format.

MOTIVATION: CRAM has established itself as a high compression alternative to the BAM file format for DNA sequencing data. We describe updates to further improve this on modern sequencing instruments. RESULTS: With Illumina data CRAM 3.1 is 7-15% smaller than the equivalent CRAM 3.0 file, and 50-70% smaller than the corresponding BAM file. Long-read technology shows more modest compression due to the presence of high-entropy signals. AVAILABILITY AND IMPLEMENTATION: The CRAM 3.0 specification is freely available from https://samtools.github.io/hts-specs/CRAMv3.pdf. The CRAM 3.1 improvements are available in a separate OpenSource HTScodecs library from https://github.com/samtools/htscodecs, and have been incorporated into HTSlib. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.