Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes

被引:49
|
作者
Seguin-Orlando, Andaine [1 ]
Schubert, Mikkel [1 ]
Clary, Joel [2 ]
Stagegaard, Julia [3 ]
Alberdi, Maria T. [4 ]
Luis Prado, Jose [5 ]
Prieto, Alfredo [6 ,7 ]
Willerslev, Eske [1 ]
Orlando, Ludovic [1 ]
机构
[1] Univ Copenhagen, Nat Hist Museum Denmark, Ctr GeoGenet, Copenhagen, Denmark
[2] Musee Confluences, Ctr Conservat & Etud Collect, Lyon, France
[3] Ebeltoft Safari, Ree Pk, Ebeltoft, Denmark
[4] CSIC, Museo Nacl Ciencias Nat, Dept Paleobiol, Madrid, Spain
[5] Univ Nacl Ctr Prov Buenos Aires, Consejo Nacl Invest Cient & Tecn, Olavarria, Argentina
[6] Univ Magallanes, Inst Patagonia, Punta Arenas, Chile
[7] Univ Autonoma Barcelona, Programa Doctorado, E-08193 Barcelona, Spain
来源
PLOS ONE | 2013年 / 8卷 / 10期
基金
新加坡国家研究基金会;
关键词
EXTINCT; DAMAGE; CAVE; PATTERNS; INSIGHTS; HISTORY; HOMININ; REVEAL;
D O I
10.1371/journal.pone.0078575
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.
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页数:11
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