Automated analysis of immunoglobulin genes from high-throughput sequencing: life without a template.

BACKGROUND: Immunoglobulin (that is, antibody) and T cell receptor genes are created through somatic gene rearrangement from gene segment libraries. Immunoglobulin genes are further diversified by somatic hypermutation and selection during the immune response. Studying the repertoires of these genes yields valuable insights into immune system function in infections, aging, autoimmune diseases and cancers. The introduction of high throughput sequencing has generated unprecedented amounts of repertoire and mutation data from immunoglobulin genes. However, common analysis programs are not appropriate for pre-processing and analyzing these data due to the lack of a template or reference for the whole gene. RESULTS: We present here the automated analysis pipeline we created for this purpose, which integrates various software packages of our own development and others', and demonstrate its performance. CONCLUSIONS: Our analysis pipeline presented here is highly modular, and makes it possible to analyze the data resulting from high-throughput sequencing of immunoglobulin genes, in spite of the lack of a template gene. An executable version of the Automation program (and its source code) is freely available for downloading from our website: http://immsilico2.lnx.biu.ac.il/Software.html.

Automated cleaning and pre-processing of immunoglobulin gene sequences from high-throughput sequencing.

High-throughput sequencing (HTS) yields tens of thousands to millions of sequences that require a large amount of pre-processing work to clean various artifacts. Such cleaning cannot be performed manually. Existing programs are not suitable for immunoglobulin (Ig) genes, which are variable and often highly mutated. This paper describes Ig High-Throughput Sequencing Cleaner (Ig-HTS-Cleaner), a program containing a simple cleaning procedure that successfully deals with pre-processing of Ig sequences derived from HTS, and Ig Insertion-Deletion Identifier (Ig-Indel-Identifier), a program for identifying legitimate and artifact insertions and/or deletions (indels). Our programs were designed for analyzing Ig gene sequences obtained by 454 sequencing, but they are applicable to all types of sequences and sequencing platforms. Ig-HTS-Cleaner and Ig-Indel-Identifier have been implemented in Java and saved as executable JAR files, supported on Linux and MS Windows. No special requirements are needed in order to run the programs, except for correctly constructing the input files as explained in the text. The programs' performance has been tested and validated on real and simulated data sets.