title: Biases in the SMART-DNA library preparation method associated with genomic poly dA/dT sequences.
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.
authors.affiliation: Department of Microbiology and Molecular Genetics, IMRIC, The Hebrew University Hadassah Medical School, Jerusalem, Israel.
authors.forename: Oriya
authors.identifier: http://orcid.org/0000-0002-7901-731X
authors.initials: O
authors.lastname: Vardi
authors.affiliation: Department of Microbiology and Molecular Genetics, IMRIC, The Hebrew University Hadassah Medical School, Jerusalem, Israel.
authors.forename: Inbal
authors.initials: I
authors.lastname: Shamir
authors.affiliation: Department of Microbiology and Molecular Genetics, IMRIC, The Hebrew University Hadassah Medical School, Jerusalem, Israel.
authors.forename: Elisheva
authors.initials: E
authors.lastname: Javasky
authors.affiliation: Department of Medicine, University of California San Diego, La Jolla, CA, United States of America.
authors.forename: Alon
authors.initials: A
authors.lastname: Goren
authors.affiliation: Department of Microbiology and Molecular Genetics, IMRIC, The Hebrew University Hadassah Medical School, Jerusalem, Israel.
authors.forename: Itamar
authors.initials: I
authors.lastname: Simon
mesh_terms: D015723:Gene Library; D016678:Genome; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D011067:Poly dA-dT / Q000235:genetics*; D012313:RNA / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D011067:Poly dA-dT; D012313:RNA