Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone

Abstract

ObjectiveA key challenge in ancient DNA research is massive microbial DNA contamination from the deposition site which accumulates post mortem in the study organism’s remains. Two simple and cost-effective methods to enrich the relative endogenous fraction of DNA in ancient samples involve treatment of sample powder with either bleach or Proteinase K pre-digestion prior to DNA extraction. Both approaches have yielded promising but varying results in other studies. Here, we contribute data on the performance of these methods using a comprehensive and systematic series of experiments applied to a single ancient bone fragment from a giant panda (Ailuropoda melanoleuca).ResultsBleach and pre-digestion treatments increased the endogenous DNA content up to ninefold. However, the absolute amount of DNA retrieved was dramatically reduced by all treatments. We also observed reduced DNA damage patterns in pre-treated libraries compared to untreated ones, resulting in longer mean fragment lengths and reduced thymine over-representation at fragment ends. Guanine–cytosine (GC) contents of both mapped and total reads are consistent between treatments and conform to general expectations, indicating no obvious biasing effect of the applied methods. Our results therefore confirm the value of bleach and pre-digestion as tools in palaeogenomic studies, providing sufficient material is available.Electronic supplementary materialThe online version of this article (10.1186/s13104-017-3061-3) contains supplementary material, which is available to authorized users.

Optimized DNA sampling of ancient bones using Computed Tomography scans

“…DNA extracts were then converted into Illumina sequencing libraries using a single‐stranded approach described in Gansauge and Meyer (), with a reduced concentration of Circligase II as described in Basler et al. (). The optimal number of library amplification PCR cycles was determined in advance using qPCR, as described in Gansauge and Meyer (), replicating the procedure reported in Basler et al.…”

“…The optimal number of library amplification PCR cycles was determined in advance using qPCR, as described in Gansauge and Meyer (), replicating the procedure reported in Basler et al. (). Indexing PCR was then performed in a reaction volume of 80 μl, using 20 μl template library, Accuprime Pfx DNA polymerase and tailed primers to generate dual‐indexed library molecules.…”

“…Such methods include predigestion with proteinase K (Damgaard et al, 2015;Gamba et al, 2016;Clio Der, Balanovsky, Templeton, & Llamas, 2014) and pretreatment with phosphate buffer or sodium hypochlorite (Korlevi c et al, 2015). However, all pretreatment approaches are associated with a parallel loss in endogenous DNA, which is frequently dramatic (Basler et al, 2017;Korlevi c et al, 2015). Sample pretreatment may also be unpredictable (Basler et al, 2017) and has even been contra-indicated for samples with very limited availability of bone material (Korlevi c et al, 2015).…”

“…However, all pretreatment approaches are associated with a parallel loss in endogenous DNA, which is frequently dramatic (Basler et al, 2017;Korlevi c et al, 2015). Sample pretreatment may also be unpredictable (Basler et al, 2017) and has even been contra-indicated for samples with very limited availability of bone material (Korlevi c et al, 2015). A third approach for increasing endogenous content is to utilize laboratory methods that target specific DNA fragment sizes, as reported for DNA extraction (Dabney et al, 2013;Glocke & Meyer, 2017) and some library preparation methods (Bennett et al, 2014).…”

Once lost, twice found: Combined analysis of ancient giant panda sequences characterises extinct clade

“…However, this represents a tiny fraction of their distribution during the Pleistocene, when they occurred across most of southern China as well as adjacent parts of Myanmar, Thailand, and Vietnam (Jablonski et al., ). Although pandas have frequently been the subject of genetic studies (Li et al., ; Zhao et al., ), until very recently, ancient DNA sequences have proved elusive (Basler et al., ), potentially reflecting advanced DNA degradation typical of ancient bones deposited in tropical and subtropical environments (Kehlmaier et al., ; Min‐Shan Ko et al., ). This year, a study by Sheng et al.…”