Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

被引:188
作者
Gansauge, Marie-Theres [1 ]
Gerber, Tobias [1 ]
Glocke, Isabelle [1 ]
Korlevic, Petra [1 ]
Lippik, Laurin [1 ]
Nagel, Sarah [1 ]
Riehl, Lara Maria [2 ]
Schmidt, Anna [1 ]
Meyer, Matthias [1 ]
机构
[1] Max Planck Inst Evolutionary Anthropol, Dept Evolutionary Genet, D-04103 Leipzig, Germany
[2] Univ Med Ctr Ulm, Dept Pediat & Adolescent Med, D-89075 Ulm, Germany
关键词
CELL-FREE DNA; GENOME SEQUENCE; ANCIENT DNA; IN-SITU; LIGATION; AMPLIFICATION; DAMAGE; NEANDERTHAL; ORIGIN;
D O I
10.1093/nar/gkx033
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页数:10
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