期刊
BRITISH JOURNAL OF HAEMATOLOGY
卷 157, 期 6, 页码 709-717出版社
WILEY
DOI: 10.1111/j.1365-2141.2012.09116.x
关键词
chronic lymphocytic leukaemia; aetiology; virus; high-throughput sequencing; digital transcriptome subtraction
类别
资金
- Agencia Nacional de Investigacion e Innovacion, Fondo Clemente Estable [FCE 2009_2611]
- Centre Nationale de la Recherche Scientifique (CNRS - LIA Laboratoire Franco-Uruguayen sur la Pathogenese virale des leucemies)
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 similar to 000 reads per sample. No viral candidate could be found. Illumina single-end sequencing for 115 cycles yielded an average of 26 +/- 2.5million filtered reads per sample, of which 2.2 +/- 0.6 similar to 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 similar to million sequences, we have not found evidence for a putative viral candidate in CLL.
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