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.

Motivation: The bulk of space taken up by NGS sequencing CRAM files consists of per-base quality values. Most of these are unnecessary for variant calling, offering an opportunity for space saving. Results: On the Syndip test set, a 17 fold reduction in the quality storage portion of a CRAM file can be achieved while maintaining variant calling accuracy. The size reduction of an entire CRAM file varied from 2.2 to 7.4 fold, depending on the non-quality content of the original file (see Supplementary Material S6 for details). Availability and implementation: Crumble is OpenSource and can be obtained from https://github.com/jkbonfield/crumble. Supplementary information: Supplementary data are available at Bioinformatics online.

Abstract Motivation: The reference CRAM file format implementation is in Java. We present ‘Scramble’: a new C implementation of SAM, BAM and CRAM file I/O. Results: The C implementation of for CRAM is 1.5–1.7× slower than BAM at decoding but 1.8–2.6× faster at encoding. We see file size savings of 34–55%. Availability and implementation: Source code is available at http://sourceforge.net/projects/staden/files/io_lib/ under the BSD software licence. Contact: [email protected] Supplementary information : Supplementary data are available at Bioinformatics online.

MOTIVATION: Existing sequence assembly editors struggle with the volumes of data now readily available from the latest generation of DNA sequencing instruments. RESULTS: We describe the Gap5 software along with the data structures and algorithms used that allow it to be scalable. We demonstrate this with an assembly of 1.1 billion sequence fragments and compare the performance with several other programs. We analyse the memory, CPU, I/O usage and file sizes used by Gap5. AVAILABILITY AND IMPLEMENTATION: Gap5 is part of the Staden Package and is available under an Open Source licence from http://staden.sourceforge.net. It is implemented in C and Tcl/Tk. Currently it works on Unix systems only.

Abstract Trev is a DNA trace editor and viewer, which is available free for UNIX and Microsoft Windows platforms. It can read all the commonly used file formats, including the new, compact ZTR files. Availability: ftp://ftp.mrc-lmb.cam.ac.uk/pub/staden/trev Contact: [email protected] * To whom correspondence should be addressed.

Abstract Motivation: To produce an open and extensible file format for DNA trace data which produces compact files suitable for large-scale storage and efficient use of internet bandwidth. Results: We have created an extensible format named ZTR. For a set of data taken from an ABI-3700 the ZTR format produces trace files which require 61.6% of the disk space used by gzipped SCFv3, and which can be written and read at greater speed. The compression algorithms used for the trace amplitudes are used within the National Center for Biotechnology Information (NCBI) trace archive. Availability: Source code is available from ftp://ftp.mrc-lmb.cam.ac.uk/pub/staden/io_lib/io_lib.tar.gz. A complete format description can be found at http://www.mrc-lmb.cam.ac.uk/pubseq/ztr.html. Test data is available from ftp://ftp.mrc-lmb.cam.ac.uk/pub/staden/io_lib/test_data. Contact: [email protected] * To whom correspondence should be addressed.