Construction of 12 Multiplex Transcriptome Libraries for Roche/454 Pyroseqeuencing Platform
Next-generation sequencing technologies have become an important tool to promote research on molecular biologic mechanism of marine organisms. In this study, 12 transcriptomice libraries,each labeled by a specific multiplex identifier (MID) of 10 nu-cleotide.were constructed from three marine species:small abalone Huliolis diversicolor,Sengmao oyster Saccostrea cucullata and unicell green algae Piatymonas suhcordiformis. To make an appropriate mixture ratio,DNA concentration of each library was determined by qPCR,Qubit and NanoDrop-1000 methods. The normalized and weighted average concentration numbers were used to pool libraries to a setting ratio. To evaluate the quality of the libraries, traditional Sanger sequencing was performed to sequence 192 cDNA fragments. Among them, 191 fragments had the required AB format on Roche/454 platform,e.g. ,454A sequence was located on one end of the cDNA fragment and 454B sequence was located on the other end. And MID barcodes could be identified from 189 cDNA fragments. The average length of the cDNA fragments was 348 bp. To further evaluate the libraries, a titrimetric sequencing was performed on a Roche/454 GSFLX-Titanium platform and 34 642 sequences were generated. MID barcodes could be identified from 32 872 sequences (94. 9%) and each of them could be precisely assigned to one of the 12 cDNA libraries. The real proportions of each MID calculated from titrimetric sequences were used to evaluate the three DNA concentration determined methods,playing an important role in library pooltng. Results showed that the qPCR method could be the most credible one. As a conclusion, the transcriptomice library construction procedure described here was very stable and reliable, and it could be widely applied on transcriptomics research.
国家科技期刊平台
NSTL
万方数据
维普
