To Clone or Not To Clone: Method Analysis for Retrieving Consensus Sequences In Ancient DNA Samples

Abstract

The challenges associated with the retrieval and authentication of ancient DNA (aDNA) evidence are principally due to post-mortem damage which makes ancient samples particularly prone to contamination from “modern” DNA sources. The necessity for authentication of results has led many aDNA researchers to adopt methods considered to be “gold standards” in the field, including cloning aDNA amplicons as opposed to directly sequencing them. However, no standardized protocol has emerged regarding the necessary number of clones to sequence, how a consensus sequence is most appropriately derived, or how results should be reported in the literature. In addition, there has been no systematic demonstration of the degree to which direct sequences are affected by damage or whether direct sequencing would provide disparate results from a consensus of clones.To address this issue, a comparative study was designed to examine both cloned and direct sequences amplified from ∼3,500 year-old ancient northern fur seal DNA extracts. Majority rules and the Consensus Confidence Program were used to generate consensus sequences for each individual from the cloned sequences, which exhibited damage at 31 of 139 base pairs across all clones. In no instance did the consensus of clones differ from the direct sequence. This study demonstrates that, when appropriate, cloning need not be the default method, but instead, should be used as a measure of authentication on a case-by-case basis, especially when this practice adds time and cost to studies where it may be superfluous.

Historically low mitochondrial DNA diversity in koalas (Phascolarctos cinereus)

“…One of the suggested criteria is sub-cloning and sequencing of multiple clones. However, when a single sequence variant is expected, as with mtDNA haplotypes, it has recently been suggested that direct sequencing may be as accurate as cloning and sequencing [29]. We compared the results of high throughput sequencing of PCR amplicons to those of direct Sanger sequencing of PCR products.…”

Recovery and amplification of ancient DNA from Herculaneum victims killed by the 79 AD Vesuvius hot surges

“…In our study we did not consider it necessary to clone amplicons because our chromatograms did not show any ambiguous nucleotide positions. On the other hand, as showed by Winters et al (2011) in their study on aDNA, the consensus sequence of clones does not differ from the direct sequence.…”

Next generation sequencing as a suitable analysis for authentication of degraded and ancient DNA

“…Although widely and long used on aDNA studies, it never has counted on a consensus as to the number of required clones or the minimum number of variations that is needed to consider a sequence as contaminated result [12,13].…”