B303: Comparison of different NGS library construction methods for single cell sequencing

Peter Hahn1,Rumeysa Akinci-Tolun1,Isabell Haupt1,Ioanna Andreou1,Christian Korfhage1,Nan Fang1

1QIAGEN GmbH, QIAGEN Strasse, Hilden, Germany

Presenting date: Tuesday 3 November

Background

 

Recent advances in whole genome and whole transcriptome amplification technologies and next generation sequencing have enabled whole genome or transcriptome sequencing at the single cell level. Single cell sequencing studies have yielded new insights into the heterogeneity of the genome and transcriptome in individual cells. Such heterogeneity at the single cell level has been shown to be closely related to cellular function, differentiation, development, and diseases.

A critical element of the single cell sequencing workflow is sequencing library construction step following whole genome or transcriptome amplification.  An efficient library construction method is required to convert a high percentage of the DNA fragments to adaptor-ligated sequencing library and ensure high sequence complexity of the library. Furthermore, uniform representation of all genomic regions in a sequencing library is essential for retaining all important sequence information.

Method

 

Here we compared two library construction methods following REPLI-g MDA-mediated whole genome amplification (WGA) or whole transcriptome amplification (WTA):  a ligation-based library construction method using GeneRead Library Prep Kit (QIAGEN); and a tagmentation-based method using Nextera DNA Sample Prep Kit (Illumina).

Results

 

Our results demonstrated that the Nextera library construction method can be directly used with the REPLI-g-amplified DNA following MDA reaction without the need for DNA purification. However, compared with the tagmentation method, the ligation-based library construction method is more flexible with the input DNA amount and delivers sequencing libraries with higher complexity and less bias, which is critical for sensitive applications such as identification of genomic variants or comprehensive profiling of transcriptome. 

Conclusion

 

The seamless procedure of directly using REPLI-g-amplified DNA as template for library construction without the need of purification could be beneficial if working with a high number of samples or if the complete workflow of single WGA/WTA and library construction should be automated.