Isolation, Library Preparation, and Bioinformatic Analysis of Historical and Ancient Plant DNA

Abstract

The ability to sequence DNA retrieved from ancient and historical material plays a crucial role in reinforcing evolutionary and anthropological inference. While the focus of the field is largely on analyzing DNA from ancient hominids and other animals, we have also learned from plant ancient DNA (aDNA), in particular, about human farming practices, crop domestication, environment management, species invasion, and adaptation to various environmental conditions. In the following protocols, we outline best practices for plant aDNA isolation, preparation for sequencing, bioinformatic processing, and authentication. We describe the process all the way from processing of archaeological or historical plant material to characterizing and authenticating sequencing reads. In alternative protocols, we include modifications to this process that are tailored to strongly degraded DNA. Throughout, we stress the importance of precautionary measures to successfully analyze aDNA. Finally, we discuss the evolution of the archaeogenomics field and the development of new methods, which both shaped this protocol.

Molecular Clocks and Archeogenomics of a Late Period Egyptian Date Palm Leaf Reveal Introgression from Wild Relatives and Add Timestamps on the Domestication

“…Read length distributions were centered on 35 bp ( supplementary fig. S1 , Supplementary Material online), consistent with sequencing data from similarly aged material ( Ramos-Madrigal et al 2016 ; Scott et al 2019 ; Latorre et al 2020 ). To assess whether these misincorporations could affect the reliability of downstream analyses involving the Saqqara leaf, we compared the Transition/Transversion (Ti/Tv) ratio and error rate (i.e., excess of derived alleles compared with a sample that is free of misincorporations; supplementary fig.…”

“…These represented 198 contigs, or 26.8% (149.01 Mb) of the P. dactylifera ’ nuclear genome assembly. We investigated the proportion and position of mis-incorporated nucleotides in the Saqqara date palm leaf DNA following the protocol of Latorre et al (2020) , that is, using aligned aDNA reads and the tool mapDamage2 v.2.0.9 ( Jónsson et al 2013 ). We compared nucleotide mis-incorporation patterns between the aligned aDNA reads of the Saqqara date palm leaf and DNA reads of modern date palm accessions (SRR5120110).…”

Revised Species Delimitation in the Giant Water Lily Genus Victoria (Nymphaeaceae) Confirms a New Species and Has Implications for Its Conservation

“…We trimmed the raw read data using AdapterRemoval v2.3.2 ( Schubert et al, 2016 ) with the ‘collapse’ option to maximize retention of shorter reads, a consideration based on our dataset having a large proportion of herbarium specimens ( Latorre et al, 2020 ). We aligned the trimmed reads to the transcriptomic reference set of reads using bwa v 0.7.17 ( Li and Durbin, 2009 ), with the mem algorithm (suited to long reads and seeds alignments with exact matches) for the samples from fresh material and aln for the herbarium material (suited to short reads and allows for mismatches).…”

The untapped potential of macrofossils in ancient plant DNA research

“…The fragmented, low‐quality, and fragile nature of plant aDNA requires strict protocols to recover generally low quantities of degraded DNA and minimize contamination from modern sources (Kistler et al ., 2020; Latorre et al ., 2020). Thus, all extraction and preamplification steps should be performed in dedicated aDNA laboratories to avoid and/or identify sources of contamination (Kistler et al ., 2020).…”

“…Thus, all extraction and preamplification steps should be performed in dedicated aDNA laboratories to avoid and/or identify sources of contamination (Kistler et al ., 2020). Different methods have been developed that optimize the recovery and processing of the short and damaged plant aDNA fragments (Latorre et al ., 2020). However, there is currently no standard protocol for DNA extraction from ancient plant remains, largely due to the diversity of plant taxa and tissue types recovered in ancient deposits (Lendvay et al ., 2018a).…”