Search for an aetiological virus candidate in chronic lymphocytic leukaemia by extensive transcriptome analysis
Search for an aetiological virus candidate in chronic lymphocytic leukaemia by extensive transcriptome analysis
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DOI：
10.1111/j.1365-2141.2012.09116.x
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发表时间：
2012-06-01
影响因子：
6.5
通讯作者：
Pritsch, Otto
中科院分区：
文献类型：
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作者：
Rego, Natalia;Bianchi, Sergio;Pritsch, Otto
作为一种确定慢性淋巴细胞白血病（CLL）病因的方法，我们通过结合高通量测序和数字减影来寻找在人类CLL B细胞中表达的病毒。采用454 Life Sciences技术对来自5名CLL患者和5名健康供体的合并B细胞mRNA转录组进行测序。通过BLAST（基本局部比对搜索工具）和BLAT（BLAST样比对工具）搜索排除人读段。用BLAST针对病毒数据库筛选剩余读段。使用Illumina技术对来自两名CLL患者的经纯化的B细胞进行测序以实现测序深度，所述经纯化的B细胞具有和不具有佛波醇酯刺激。Burrows-Wheeler Aligner作图和BLAST搜索用于Illumina数据。焦磷酸测序导致每个样品约400个类似于1000个读数。Illumina单端测序115个循环，每个样本平均产生26 +/-250万个过滤读数，其中2.2 +/- 0.6类似于100万个未映射到人类参考。在佛波酯刺激后的一个样品中，针对病毒和人类数据库对这些读段进行BLAST搜索，将9个读段分配给Epstein-Barr病毒来源。其他显示推定病毒来源的读数在进一步分析后被排除。尽管对CLL转录组的深入分析达到了超过100个类似于百万个序列，但我们尚未发现CLL中推定的病毒候选物的证据。
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.