DOI: 10.5061/dryad.kprr4xhd6
title: Hiding in plain sight: The biomolecular identification of pinniped use in medieval manuscripts – MALDI and mtDNA data set

description: Medieval manuscripts still in their original bindings are rare. Taking
 advantage of the diversity of bindings in Cistercian libraries during the
 12th and 13th centuries, this study focuses on the biocodicological
 analysis of medieval bindings, with particular emphasis on the use of
 sealskins. Using innovative methods such as eZooMS and aDNA analysis, this
 research identifies the species and origin of the leather used as pinniped
 (seal) species. In particular, the collagen-based eZooMS technique
 facilitated the classification of seven chemises into the pinniped clade,
 although species identification remained elusive, with one exception. aDNA
 analysis was instrumental in verifying the origin of the sealskins, with
 most samples matching harbour and harp seals and sourced to populations in
 Scandinavia, Scotland, and Iceland or Greenland. This supports the notion
 of a robust medieval trade network that went well beyond local sourcing,
 linking the Cistercians to wider economic circuits that included fur trade
 with the Norse. The study highlights the use of an unexpected skin (seal)
 from an unexpected source (the northwestern Atlantic). The widespread use
 of sealskins in Cistercian libraries during the 12th and 13th centuries
 hints at broader trade networks which brought, for example, walrus ivory
 from the far north into continental Europe.
description: Samples An initial focus was placed on
 manuscripts bound with leather with hairs on, all dated from 1135 to 1250,
 19 from the collection of Clairvaux manuscripts, and three from other
 Cistercian collections, Clairmarais and Vauclair, enhancing the
 understanding of material composition and historical provenance across
 these collections. Sampling was conducted on the flesh
 side of the leather to avoid contamination from keratin, ensuring only
 collagen fibres were collected. A representative set of nine manuscripts
 was selected for eZooMS analysis (including six from Clairvaux),
 increasing the accuracy of visual identification techniques for the
 broader collection. Subsequently, seven of these samples were sequenced to
 provide a comparative analysis [Table 3].  
 
 Sample number 
 Call number 
 ID technique 
 EL07 Troyes
 5 eZooMS 
 EL53 
 Troyes 31  
 eZooMS, aDNA  
 EL51 Troyes
 35  eZooMS, aDNA 
 EL52
 Troyes 40,7  
 eZooMS, aDNA  
 EL55 
 Troyes 40,8  eZooMS,
 aDNA  
 EL54 Troyes
 43,3  eZooMS, aDNA 
 EL57
 Saint-Omer 701  
 eZooMS, aDNA  
 EL58 
 Saint-Omer, ms 37  
 eZooMS, aDNA  
 Laon01 Ms
 176  eZooMS 
 Laon02
 Ms 8bis  
 eZooMS   
 Laon03 Laon
 166  eZooMS 
 
   Protein analysis
 Samples were collected using PVC erasers, following the procedure
 outlined in Fiddyment et al. 2015. Using the established eZooMS technique,
 samples were first processed with MALDI-TOF MS. The choice of eZooMS is
 justified by the heritage nature of the collection studied, which requires
 the use of non-destructive (or micro-invasive) analysis techniques. It
 also has the advantage of not requiring transportation of the manuscripts
 from their current storage. DNA analysis
 Extraction DNA was extracted from eraser
 crumb samples taken from each document following the protocol of Teasdale
 et al, in a dedicated ancient DNA laboratory at BioArCh, University of
 York. Illumina sequencing libraries were prepared for each sample
 following the protocol of Meyer and Kircher as modified by Gamba.
 Libraries were then sequenced on two lanes of a HiSeq4000 at the
 University of Copenhagen.  Sequencing and
 bioinformatics The two FastQ files for each sample
 were first quality checked using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and trimmed for adapter sequences using cutadapt. Sequences were first aligned to multiple reference genomes using FastQ Screen (supplementary table 1) and then to the predicted source species using BWA (using recommended settings for aDNA. Alignments were then filtered for PCR duplicates and low mapping quality reads using SAMtools, quality controlled using Qualimap 2 and visualised in IGV (Integrative genomics viewer). Final fasta consensus sequences for each sample were produced using angsd and visualised using Seaview. Haplotype network and phylogenetic analysis The likely geographical origin of the four harbour seal binders was inferred through construction of a median-joining haplotype network using PopART and a reference dataset of 954 mtDNA haplotypes (422 bp) from contemporary North Atlantic harbour seals compiled from previous studies, and unpublished data (Olsen MT). The phylogenetic analysis on sample EL58, identified as harp seal, was carried out in MEGA7. EL58 was aligned with 53 contemporary North Atlantic harp seal mitochondrial genomes. In MEGA, the alignment was conducted using MUSCLE, and a Maximum Likelihood phylogeny was constructed based on the Hasegawa-Kishino-Yano model with 1000 bootstrap repetitions. Metagenomic Profiling Metagenomic profiling was undertaken on adapter-trimmed and de-duplicated reads by k-mer alignment, using the ConClave sorting method to resolve multi-mapping reads from KMA following the approach in CCMetagen. This used the NCBI nt database as a reference dataset, without unclassified environmental sequences. CCMetagen was found to produce a significantly reduced rate of false positives in comparison with other k-mer based classification methods. KMA was run with the following options in effect: “kma -1t1 -mem_mode -and -apm f”. 
description: # MALDI and mtDNA data set
 [https://doi.org/10.5061/dryad.kprr4xhd6](https://doi.org/10.5061/dryad.kprr4xhd6) ### RSOS-241090\_eZooMS\_data.zip The folder contains MALDI-TOF data exported in txt format from eZooMS analysis of tawed skin samples. The first column indicates m/z and the second column indicates intensity. List of the samples analysed with eZooMS (MALDI) and aDNA (mtDNA) of manuscripts from the abbeys of Clairvaux (Troyes library), Clairmarais (St Omer library) and Vauclair (Laon Library). | Sample number | Call number | ID technique | | :------------ | :----------------- | :------------ | | EL07 | Troyes 5 | eZooMS | | EL53 | Troyes 31  | eZooMS, aDNA  | | EL51 | Troyes 35  | eZooMS, aDNA  | | EL52 | Troyes 40,7  | eZooMS, aDNA  | | EL55 | Troyes 40,8  | eZooMS, aDNA  | | EL54 | Troyes 43,3  | eZooMS, aDNA  | | EL57 | Saint-Omer 701  | eZooMS, aDNA  | | EL58 | Saint-Omer, ms 37  | eZooMS, aDNA  | | Laon01 | Ms 176  | eZooMS  | | Laon02 | Ms 8bis  | eZooMS   | | Laon03 | Laon 166  | eZooMS  | ### seal\_all\_mtDNA.fa consensus DNA sequences in fasta format for the samples used in the phylogenetic analysis presented in this study. ### krona visualizations (Zenodo) Krona visualization of the metagenomic data recovered from each sample presented in this study. ### FastQ files * PRI_LT7L_EL51_S10_L003_R1_001_cutadapt.clean.fastq.gz - Sequencing data for sample EL51 in fastq format adapter trimmed and host sequences removed.  * PRI_LT7L_EL52_S11_L003_R1_001_cutadapt.clean.fastq.gz - Sequencing data for sample EL52 in fastq format adapter trimmed and host sequences removed. * PRI_LT7L_EL53_S12_L003_R1_001_cutadapt.clean.fastq.gz - Sequencing data for sample EL53 in fastq format adapter trimmed and host sequences removed. * PRI_LT7L_EL54_S13_L003_R1_001_cutadapt.clean.fastq.gz - Sequencing data for sample EL54 in fastq format adapter trimmed and host sequences removed. * PRI_LT7L_EL55_S14_L003_R1_001_cutadapt.clean.fastq.gz - Sequencing data for sample EL55 in fastq format adapter trimmed and host sequences removed. * PRI_LT7L_EL57_S15_L003_R1_001_cutadapt.clean.fastq.gz - Sequencing data for sample EL57 in fastq format adapter trimmed and host sequences removed. * PRI_LT7L_EL58_S16_L003_R1_001_cutadapt.clean.fastq.gz - Sequencing data for sample EL58 in fastq format adapter trimmed and host sequences removed.