Journal
BMC GENOMICS
Volume 15, Issue -, Pages -Publisher
BIOMED CENTRAL LTD
DOI: 10.1186/1471-2164-15-569
Keywords
Next-generation sequencing; Bias; Ion Torrent; Mth K97A; Thermostable; CircLigase; Small RNA
Funding
- Biotechnology and Biological Sciences Research Council [BB/D526653/1]
- BBSRC Professorial Fellowship [BB/F02326X/2]
- Medical Research Council
- BBSRC [BB/F02326X/2, BB/F000065/1] Funding Source: UKRI
- MRC [MC_UP_A600_1023] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/F02326X/2, BB/F000065/1] Funding Source: researchfish
- Medical Research Council [MC_UP_A600_1023] Funding Source: researchfish
Ask authors/readers for more resources
Background: Next-generation sequencing does not yield fully unbiased estimates for read abundance, which may impact on the conclusions that can be drawn from sequencing data. The ligation step in RNA sequencing library generation is a known source of bias, motivating developments in enzyme technology and library construction protocols. We present the first comparison of the standard duplex adaptor protocol supplied by Life Technologies for use on the Ion Torrent PGM with an alternate single adaptor approach involving CircLigase (CircLig protocol). A correlation between over-representation in sequenced libraries and degree of secondary structure has been reported previously, therefore we also investigated whether bias could be reduced by ligation with an enzyme that functions at a temperature not permissive for such structure. Results: A pool of small RNA fragments of known composition was converted into a sequencing library using one of three protocols and sequenced on an Ion Torrent PGM. The CircLig protocol resulted in less over-representation of specific sequences than the standard protocol. Over-represented sequences are more likely to be predicted to have secondary structure and to co-fold with adaptor sequences. However, use of the thermostable ligase Methanobacterium thermoautotrophicum RNA ligase K97A (Mth K97A) was not sufficient to reduce bias. Conclusions: The single adaptor CircLigase-based approach significantly reduces, but does not eliminate, bias in Ion Torrent data. Ligases that function at temperatures to remove the possible influence of secondary structure on library generation may be of value, although Mth K97A is not effective in this case.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available