title: Search for an aetiological virus candidate in chronic lymphocytic leukaemia by extensive transcriptome analysis.
abstract: As an approach to determining the aetiology of chronic lymphocytic leukaemia (CLL), we searched for a virus expressed in human CLL B-cells by combining high-throughput sequencing and digital subtraction. Pooled B-cell mRNA transcriptomes from five CLL patients and five healthy donors were sequenced with 454 Life Sciences technology. Human reads were excluded by BLAST (Basic Local Alignment Search Tool) and BLAT (BLAST-like alignment tool) searches. Remaining reads were screened with BLAST against viral databases. Purified B-cells from two CLL patients, with and without stimulation by phorbol-esters, were sequenced using Illumina technology to achieve depth of sequencing. Burrows-Wheeler Aligner mapping and BLAST searches were used for the Illumina data. Pyrosequencing resulted in about 400 000 reads per sample. No viral candidate could be found. Illumina single-end sequencing for 115 cycles yielded an average of 26 ± 2·5 million filtered reads per sample, of which 2·2 ± 0·6 million remained unmapped to human references. BLAST searches of these reads against viral and human databases assigned nine reads to an Epstein-Barr virus origin, in one sample following phorbol-ester stimulation. Other reads showing a putative viral origin were dismissed after further analysis. Despite an in-depth analysis of the CLL transcriptome reaching more than 100 million sequences, we have not found evidence for a putative viral candidate in CLL.
authors.affiliation: Institut Pasteur de Montevideo, Montevideo, Uruguay.
authors.forename: Natalia
authors.initials: N
authors.lastname: Rego
authors.forename: Sergio
authors.initials: S
authors.lastname: Bianchi
authors.forename: Pilar
authors.initials: P
authors.lastname: Moreno
authors.forename: Helena
authors.initials: H
authors.lastname: Persson
authors.forename: Anders
authors.initials: A
authors.lastname: Kvist
authors.forename: Alvaro
authors.initials: A
authors.lastname: Pena
authors.forename: Pablo
authors.initials: P
authors.lastname: Oppezzo
authors.forename: Hugo
authors.initials: H
authors.lastname: Naya
authors.forename: Carlos
authors.initials: C
authors.lastname: Rovira
authors.forename: Guillermo
authors.initials: G
authors.lastname: Dighiero
authors.forename: Otto
authors.initials: O
authors.lastname: Pritsch
mesh_terms: D000368:Aged; D001402:B-Lymphocytes / Q000378:metabolism / Q000821:virology*; D005260:Female; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics* / Q000378:metabolism / Q000821:virology*; D008297:Male; D008875:Middle Aged; D059467:Transcriptome