DOI: 10.5061/dryad.vq83bk3qj
title: Data from: Proportion methylation at a set of CpGs from 4 amplicons

description: The age structure of populations, or the ageing rate of individuals,
 impacts aspects of animal ecology, epidemiology and conservation. Yet for
 many wild organisms, age is an inaccessible trait. In many cases measuring
 age or ageing rates in the wild requires molecular biomarkers of age.
 Epigenetic clocks based on DNA methylation have been shown to accurately
 estimate the age of humans and laboratory mice, but they also show
 variable ticking rates that are associated with mortality risk above and
 beyond that predicted by chronological age. Thus, epigenetic clocks are
 proving to be useful markers of both chronological and biological age, and
 they are beginning to be applied to wild mammals and birds. We have
 acquired strong evidence that an accurate clock will be possible for the
 wood mouse Apodemus sylvaticus by adapting epigenetic information from the
 laboratory mouse. Apodemus sylvaticus is a well-studied field system that
 is amenable to experimental perturbations and longitudinal sampling of
 individuals across their lives, and these features of the wood mouse offer
 opportunities to disentangle causal relationships between ageing rates and
 environmental stress. Our wood mouse epigenetic clock is PCR-based, and so
 requires tiny amounts of tissue and non-destructive sampling. We
 quantified methylation using Oxford Nanopore sequencing technology and
 present a new bioinformatics pipeline for data analysis. We thus describe
 a new and generalizable system that should enable ecologists and other
 field biologists to go from tiny tissue samples to an epigenetic clock for
 their study animal.   
description: DNA was
 extracted using a phenol-chloroform method from ear punches taken from
 individuals of known age and kept under identical conditions in a wood
 mouse colony at the University of Edinburgh. We obtained DNA from 48 mice
 spanning from 88 to 496 days old. This slightly unusual choice of ages
 arises because we utilised mice that were part of other experiments, thus
 increasing the information gained from each animal sacrificed. Lifespan in
 wild mice is not known for certain, and many will die from extrinsic
 mortality (e.g. predation), but in our own field work we recapture 10-20%
 of tagged mice the following year, meaning wild mice may often live for
 hundreds of days. We used the
 polymerase chain reaction (PCR) to identify CpG sites in <i>Apodemus
 sylvaticus </i>that could contribute to an methylation-based
 epigenetic clock. We first bisulphite treated the DNA using the ZYMO-Gold
 DNA bisulphite conversion kit. Bisulphite treatment converts unmethylated
 cytocine to uracil, which are then converted to thymine during PCR.
 Methylated cytosines are unchanged by the treatment. For PCR of
 bisulphite-converted DNA, we focused initially on five genes studied by
 Han <i>et al </i>(<i>8</i>): <i>Prima1,
 HSP4, Kcns1, Gm9312 </i>and <i>Gm7325. </i>From
 <i>Ensemble</i> we obtained <i>Mus musclus </i>DNA
 sequence that included the key sites identified by Han et al<i>
 </i>(<i>8</i>). We blasted 200-300bp of <i>Mus
 </i>sequence for each gene this into the <i>Apodemus
 sylvaticus </i>whole genome shotgun sequence available on
 <i>NCBI </i>(taxonID: 40375) to retrieve the homologous wood
 mouse sequence. We then designed <i>Apodemus-</i>specific
 primers for each gene in approximately the same location as that used by
 Han <i>et al</i> (<i>8</i>). In some cases,
 <i>Apodemus </i>contained<i> </i>a<i>
 </i>CpG<i> </i>at<i> </i>the desired primer
 location, but it was usually possible to design primers slightly up or
 downstream. In two cases, however, we inserted an ambiguous base pair at a
 CpG location<i>. </i> We used an Oxford
 Nanopore Flongle to sequence our amplicons and determine the % methylation
 at all CpG sites (Figure 1). The Flongle sequences up to ~ 1GB of DNA, but
 given the short length of our amplicons this provides considerable depth
 of coverage. Our molecular methods follow Quick et al 2017
 (<i>18</i>), with minor modifications. Amplicons were pooled
 by mouse and quantified with a Qubit, the aim being to normalise DNA
 quantities so that ~ 200 fmol of DNA were loaded onto the Flongle.
 Otherwise, the minion was run according to the manufacturer’s instructions
 (Ligation Sequencing Kit, SQK-LSK109). We developed the
 custom software `Paramether` on a Snakemake(<i>20</i>)
 framework for easy-to-use estimation of CpG methylation from nanopore
 sequences. Paramether first bins the sequences by barcode using Porechop
 (https://github.com/rrwick/Porechop). We curated a database of reference
 genes representing the bisulphite-treated, amplified gene sequences.
 Paramether queries this database using parasail
 (https://github.com/jeffdaily/parasail.git) in order to classify which
 gene each read corresponds to. Using the alignment with the reference
 genes, key sites are identified in the read and `Paramether` determines if
 a site is modified (i.e. a C or a T) or not. This process is continued for
 each read and counts of methylation are output per gene per barcode.
 Proportion methylated
 Cs at each of the 85 CpG in the sequences was studied using penalised
 regression to identify sites that correlate with age. The was achieved
 with the <i>GLMNET </i>package in R (<i>19</i>)
 using a LASSO model (mixing parameter alpha=1)  and leave-one-out cross
 validation (nfolds=nrow). The code for this is presented in the
 supplementary data.  
description: The file cpg_wide_clock.csv is the proportion methylation at each
 CpG. Rows or columns with missing values have been removed because GLMnet
 does not accept them. The file cpg_counts.csv provides information on the
 number of C's and T's counted, and thus also depth of coverage
 at each CpG.