Absolute quantitative whole transcriptome sequencing uses digital tag (UMI) technology to trace each library fragment and achieve absolute, digital and precise quantification of mRNA, lncRNA and miRNA.

True quantitative sequences are all labeled with unique UMIs, eliminating PCR amplification bias and truly reflecting the abundance of transcripts in the sample.

qPCR verification rate over 99%

True sequence The authenticity of the transcript is ensured by correcting similar sequences with the same UMI marker.

The PCR step affects the accuracy of the results during library amplification in transcriptome sequencing. Due to the PCR amplification preference, the reverse transcribed DNA sequence cannot be expanded in the same proportion during conventional transcriptome sequencing library construction, and the quantitative results are inconsistent with the starting abundance of the transcripts in the sample, affecting the accuracy of the quantitative results. The absolute quantitative transcriptome sequencing launched by Bena Technology uses UMI labeling technology to label each reverse transcribed cDNA sequence with UMI before library amplification, eliminating the interference of PCR amplification preference and truly reflecting the abundance of transcripts in the sample.

UMI will accompany the entire process of fragment amplification, sequencing, and analysis. The products amplified by PCR of the same fragment carry the same UMI. After sequencing, the UMI is used to trace the source of each fragment. By merging fragments from the same source (with the same sequence and UMI), PCR amplification duplications can be removed and the original state of the sample before amplification can be accurately restored. Errors in simultaneous amplification and sequencing will cause the same UMI to correspond to multiple different sequences. By comparing the similarities of these sequences, they can be corrected to ensure true transcripts.

48 differentially expressed genes were selected and verified by qPCR. The left figure is a correlation analysis of the results of ordinary RNA-seq and qPCR experiments, and the right figure is a correlation analysis of the full quantitative transcriptome and qPCR experiments. The absolute quantitative sequencing qPCR verification rate can reach more than 99%.

References

Shiroguchi K, et al. Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes. Proc Natl Acad Sci U S A. 2012 Jan 24;109(4):1347-52

Kivioja T, et al. Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods. 2011, 9(1):72-4