A novel method to assess the integrity of frozen archival DNA samples: Alpha-diversity ratios of short and long-read 16S rRNA gene sequences

Archival DNA samples collected and analysed for a range of research and applied questions have accumulated in the laboratories of universities, government agencies, and commercial service providers for decades. These DNA archives represent a valuable, yet largely untapped repository of genomic information. With lowering costs of, and increasing access to, high-throughput sequencing, we predict an increase in retrospective research to explore the wealth of information that resides in these archival samples. However, for this to occur, we need confidence in the integrity of the DNA samples, often stored under sub-optimal conditions and their fitness of purpose for downstream genomic analysis. Here, we borrow from a well-established concept in ancient DNA to evaluate sample integrity, defined as loss of information content in recovered amplicons, of frozen DNA samples and based on the ratio of ⍺-diversity of short and long-read 16S rRNA gene sequences. The 16S rRNA variable region of eight..., Data analysis The Pacific Biosciences Nextflow pipeline (https://github.com/PacificBiosciences/pb-16S-nf) was followed for initial data processing. Raw reads were processed, including demultiplexing by “q2-demux” in QIIME2, and quality control was assessed with q2-cutadapt. Quantitative Insights Into Microbial Ecology 2 (QIIME2 v. 2018.11) software was used to analyse the trimmed reorientated sequences (Bolyen et al., 2019). The DADA2 denoising option (Callahan et al., 2016) was selected to pick up the representative reads for generating an amplicon sequence variants (ASVs) table. ASVs generated from DADA2 were classified using the Naive Bayes classifier and SILVA reference database version 138.1 (Quast et al., 2013). For analysis between the platforms the feature table of each platform was merged, as were the representative sequences post-DADA2 denoising with QIIME2 before building the phylogenetic tree and assigning taxonomy.
Taxonomic diversity analysis All analysis was conducted wit..., , # **A novel method to assess the integrity of frozen archival DNA samples: Alpha-diversity ratios of short and long-read 16S rRNA gene sequences**

[https://doi.org/10.5061/dryad.v9s4mw73t](https://doi.org/10.5061/dryad.v9s4mw73t)

We utilized DNA extracted from various agricultural soils that were stored at -20°C in a gene bank freezer room over 20 years by the South Australian Research and Development Institute (SARDI). This DNA was collected through the PREDICTA® B DNA-based soil disease testing service for broadacre farming (PREDICTA® B). We selected 87 soil DNA extracts from three Australian states (regions), spanning 10 distinct time bins between 2001 and 2020. Our primary concern was the potential DNA degradation in the oldest samples. Therefore, we included samples from the first four years (2001-2004) and selected samples more sporadically from subsequent years (2005 onwards). Alpha-diversity ratios, using Shannon's diversity index, were calculated to determine if there was a d...