The tssv program does targeted characterisation of short structural variation. See the help (-h) of this program for a full description of the parameters.

Paired-end read support

With paired-end data in which the insert size was less than twice the read length, the two reads of each pair may overlap partially or entirely. In this setting, it is possible to use a read merging tool to combine the two reads of each pair, possibly giving rise to a longer combined read. The combined read may contain a known allele that does not fit completely in a single read.

Because repetitive sequences like STRs may perfectly align in many ways, such tools will be unable to detect whether the combined read actually contains all repeats that were originally present in the DNA molecule. If the molecule was too long, the two reads may not overlap at all; however, with a highly repetitive sequence, they may still appear to overlap significantly. It is only certain that all repeats have been captured if all of them fitted on a single read - but the flanking sequences may not be present in their entirety for such long alleles. Merging the two reads together may still yield the complete flanking sequence, allowing the allele to be detected by TSSV.

To make sure that any potentially truncated sequences are rejected, the combined reads can be provided to TSSV with upper case letters for the overlapped part (i.e., the middle, where both reads in the pair overlap) and lower case letters for the non-overlapping parts (i.e., those parts only present in one of the two reads). If TSSV aligns a flanking sequence to a completely lower case part of the combined read, this means that the flanking sequence was only present in one of the two reads and had fallen off the end of the other. In this case, it is impossible to tell whether the sequence between the flanking sequences contains all repeats that were originally present in the DNA molecule and therefore it is not meaningful to perform a regular expression match against it.

One particular paired-end read merging tool that supports this kind of output is a fork of FLASH 1.2.11 that was specifically made for this purpose, which is hosted on GitHub. This fork introduces an optional command-line argument --lowercase-overhang that, when specified, enables output compatible with the paired-end read support of TSSV.

Cheat sheet: Interpretation of letter case in TSSV input and output

Letter case in the library file:

  • All sequences in the library file should be in upper case completely. Regular expressions containing lower case letters will never match. Lower case letters in flanking sequences will always count as a mismatch in alignments.

Interpretation of letter case in input sequences:

  • Completely upper case reads are processed normally.
  • TSSV will be unable to detect any flanking sequences in lower case sequences.
  • In mixed case sequences, the regions to which the flanking sequences are aligned should contain at least one upper case letter. When a flanking sequence aligns to a completely lower case region, the match is rejected. When a pair of flanking sequences is found in the read (and neither match is rejected), the corresponding regular expression is matched case insensitively to the sequence between the flanks.

Letter case in output files:

  • Sequences in output FASTA files have the same case as the corresponding input sequences.
  • Sequences in output CSV files will be completely in upper case.

Implementation notes

TSSV will do all alignments and pattern matching against an explicitly upper case copy of each read. The library should therefore contain only upper case sequences. When a flanking sequence (as provided in the library file) has been detected in a read, TSSV will check whether the section of the read that corresponds to this flanking sequence contains any upper case letters. If this section of the read was completely lower case, the match is rejected.


Seyed Yahya Anvar, Kristiaan J. van der Gaag, Jaap W. F. van der Heijden, Marcel H. A. M. Veltrop, Rolf H. A. M. Vossen, Rick H. de Leeuw, Cor Breukel, Henk P. J. Buermans, J. Sjef Verbeek, Peter de Knijff, Johan T. den Dunnen, and Jeroen F. J. Laros, TSSV: a tool for characterization of complex allelic variants in pure and mixed genomes. Bioinformatics, first published online February 13, 2014. doi:10.1093/bioinformatics/btu068