Abstract
Avoiding biases in next generation sequencing (NGS) library preparation is crucial for obtaining reliable sequencing data. Recently, a new library preparation method has been introduced which has eliminated the need for the ligation step. This method, termed SMART (switching mechanism at the 5′ end of the RNA transcript), is based on template switching reverse transcription. To date, there has been no systematic analysis of the additional biases introduced by this method. We analysed the genomic distribution of sequenced reads prepared from genomic DNA using the SMART methodology and found a strong bias toward long (≥12bp) poly dA/dT containing genomic loci. This bias is unique to the SMART-based library preparation and does not appear when libraries are prepared with conventional ligation based methods. Although this bias is obvious only when performing paired end sequencing, it affects single end sequenced samples as well. Our analysis demonstrates that sequenced reads originating from SMART-DNA libraries are heavily skewed toward genomic poly dA/dT tracts. This bias needs to be considered when deciding to use SMART based technology for library preparation.
Highlights
Generation sequencing (NGS) technologies have become a major research tool in all fields of biology [1]
Biases in the SMART-DNA library preparation method sequencing of the ligation mediated libraries initiates from primers that recognize sequences at the end of the adapters
Bias toward poly dA/dT genomic tracts. These results suggest that the SMART- DNA library preparation methodology has a bias toward poly dA/dT tracts
Summary
Generation sequencing (NGS) technologies have become a major research tool in all fields of biology [1]. Massive sequencing is important for many fields including comparative genomics [2,3,4], human population genetics, personalized medicine [5, 6] and microbiome research [7]. New applications for NGS are constantly being developed, expanding the ability to measure all kinds of genomic and transcriptomic features [8]. All these applications include the addition of known sequences to both ends of the nucleic acid material (DNA or RNA) that are used both for amplification and for sequencing.
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