Biases in the SMART-DNA library preparation method associated with genomic poly dT sequences. Homo sapiens strain:HCT116 and HeLa-S3

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 analyzed the genomic distribution of sequence 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 also affects single end sequenced samples and thus should be taken into account in all SMART based DNA sequencing projects. We propose both computational and experimental solutions to this bias.