pastForward: a Snakemake pipeline for ancient and historical DNA with eukaryote-wide taxonomic screening and tracking of copy-number variation

Ancient and historical DNA has the potential to resolve many open questions in biology. While pipelines for processing ancient and historical DNA exist, none combine user-friendly, configurable processing with copy number variation tracking and targeted taxonomic profiling. Therefore, we developed pastForward, a fully automated Snakemake pipeline that integrates all analysis steps from raw reads to damage-rescaled BAM files in a single reproducible workflow. It performs ancient and historical DNA processing, including adapter trimming, read merging, deduplication, damage assessment, quality rescaling, and generates interactive reports summarizing the endogenous read content, library complexity, and breadth and depth coverage statistics. These reports allow users to rapidly assess the quality of sequencing data. It handles single- and paired-end NGS libraries. Mapping to multiple reference sequences is supported, facilitating co-analysis of host and endosymbiont sequences and genotyping of marker genes such as COI.

pastForward further integrates two novel tools. ECMSD (Efficient Comprehensive Mitochondrial Sequence Detector) screens each library for eukaryotic DNA by aligning reads against a mitochondrial reference database. The presence of bacteria, archaea and viruses is detected in parallel with Centrifuge. REVEAL (Read-based Estimation and visualization of Element Abundance and Loci) quantifies and visualizes copy number variation of genetic features, such as transposable elements (TEs) or gene duplications.

Two case studies demonstrate the usage of the pipeline. Using pastForward on dog genomic time series, including Neolithic samples, we confirm that the copy number of AMY2B , which encodes the starch-digesting enzyme amylase, increased during domestication. From historical D. melanogaster genomes, we recover the recent invasion of the transposable element opus . It is absent in specimens from the 1800s and present from 1933 onward. By efficiently processing large numbers of samples, pastForward facilitates longitudinal tracking of genomic features in diverse species.