Gene expression profiling by massively parallel sequencing
Gene expression profiling by massively parallel sequencing
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DOI：
10.1101/gr.6984908
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发表时间：
2008-01-01
期刊：
影响因子：
7
通讯作者：
Schlotterer, Christian
中科院分区：
文献类型：
--
作者：
Torres, Tatiana Teixeira;Metta, Muralidhar;Schlotterer, Christian
大规模平行的测序对表达分析具有巨大的希望，因为它结合了SAGE的高通量与EST测序的准确性。然而，直到现在，只有非常有限的信息可用于满足当前技术的适用性。在这里，我们评估了454个测序技术对使用果蝇的表达分析的潜力。我们表明，在454个序列读取中，短（<-80 bp）和长（>类似于300-400 bp）的cDNA片段的代表性不足。然而，可以使用雾化产生的3'cDNA片段的测序来克服454个测序技术的长度偏差。通过限制分析和雾化产生的基因表达测量值在80至300 bp范围内的片段显示出类似于复制微阵列实验的相关性（0.83-0.91）； 97％的cDNA片段可以明确映射到基因组DNA，这表明较长序列读取的优势。我们的分析表明，4,54技术具有表达分析的巨大潜力，高映射精度表明应该可以比较跨物种的表达谱。
Massively parallel sequencing holds great promise for expression profiling, as it combines the high throughput of SAGE with the accuracy of EST sequencing. Nevertheless, until now only very limited information had been available on the suitability of the Current technology to meet the requirements. Here, we evaluate the potential of 454 sequencing technology for expression profiling using Drosophila melanogaster. We show that short (< -80 bp) and long (> similar to 300-400 bp) cDNA fragments are Under-represented in 454 sequence reads. Nevertheless, sequencing of 3' cDNA fragments generated by nebulization could be used to overcome the length bias of the 454 sequencing technology. Gene expression measurements generated by restriction analysis and nebulization for fragments within the 80- to 300-bp range showed correlations similar to those reported for replicated microarray experiments (0.83-0.91); 97% of the cDNA fragments could be unambiguously mapped to the genomic DNA, demonstrating the advantage of longer Sequence reads. Our analyses suggest that the 4,54 technology has a large potential for expression profiling, and the high mapping accuracy indicates that it should be possible to compare expression profiles across species.