A Comparative Study Of Two Methods Of Transcription Library Construction

As transcriptomic,proteomics,and metabolomics constantly emerging,biology study has entered the post-genome era.The transcriptome sequencing developed as one of the first popular technology in post-genome era study has been widely used.The Central Dogma tells us that for the most organisms,the transmission of genetic information is from DNA to RNA and RNA into protein.The process of genetic information transmission from DNA to RNA is called transcription.Transcriptome analysis is vital for understanding genome function components and molecules in cells and tissues.It can also reveal the mechanism in the process of biological processes and diseases.With the development of sequencing technologies,we are now able to carry out a more in-depth on the transcriptome sequencing,so that we can find more reliable and novel transcripts.Sequencing platform has been updated quickly as well,which requires us to update the transcriptome research methods to cater to the good sequencing platform..This study selected the UHRR(Universal Hhuman Reference RNA)as experiment material.We used two methods of the transcriptome library preparation protocols(Short Insert Fragment Method,Long Insert Fragment Method)and constructed two kinds of Hiseq sequencing libraries with insert fragment length 160bp(TUHRR90)and 300bp(NUHRR150)separately.Then TUHRR90 was sequenced on Illumina Hiseq2000 sequencer using PE90 sequencing strategy.Meanwhile,NUHRR150 was run on Illumina Hiseq4000 sequencer to generated PE150 reads.A total of 16 Gb of data was obtained.QPCR quantitative results of the 1000 genes of UHRR were downloaded from supplier Website as the reference data for gene expression quantification.The data of the 300bp(NUHRR150)library was trimmed using SOAPnuke software to simulate another set of NUHRR150 data with PE90 reads.Then BGI transcriptome standard information analysis pipeline RNA_RNAref_version5.0_beta was employed to analyze TUHRR90,NUHRR90 and NUHRR150.The analysis results were compared among those data sets,such as quality of sequencing,data output,random distribution,Alternative splicing,junction discovery.And we further evaluated the gene quantitative consistency among three methods and finally compared the gene expression results to those obtained by qPCR.Analysis results showed that high throughput sequencing platform Illumina Hiseq4000 sequencing machine can provide long enough,accurate reads and can meet the needs of the analysis of the transcriptome study.Long insert fragments of the transcriptome library(300bp)generated with our modified method can be run on Illumina Hiseq4000 sequencing machine platform to obtain PE150 reads.And data production rate,quality can meet the requirements of the transcriptome analysis.Longer insert size combined with longer reads suggested the advantages in finding more alternative splicing events,junction,and gene fusion detection.The gene quantification accuracy is similar among the three data sets.Long insert transcriptome library sequence on Hiseq4000 to achieve PE150 will improve the gene structure variation detection.Thus it may replace the standard protocol using right now.