title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase.
abstract: DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3΄ biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude.
authors.affiliation: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany.
authors.forename: Marie-Theres
authors.initials: MT
authors.lastname: Gansauge
authors.affiliation: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Gerber
authors.affiliation: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Glocke
authors.affiliation: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Korlevic
authors.affiliation: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany.
authors.forename: Laurin
authors.initials: L
authors.lastname: Lippik
authors.affiliation: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Nagel
authors.affiliation: Department of Pediatrics and Adolescent Medicine, University Medical Center Ulm, 89075 Ulm, Germany.
authors.forename: Lara Maria
authors.initials: LM
authors.lastname: Riehl
authors.affiliation: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Schmidt
authors.affiliation: Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Meyer
mesh_terms: D000818:Animals; D001842:Bone and Bones / Q000737:chemistry; D004247:DNA / Q000235:genetics / Q000378:metabolism; D011088:DNA Ligases / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics* / Q000378:metabolism; D005580:Fossils; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D006736:Horses; D006801:Humans; D008099:Liver / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013552:Swine
chemical_list: D017931:DNA Primers; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D004247:DNA; D011088:DNA Ligases