Miseq platform

Two additional data sets were included for comparative purposes. First, DNA sequences from the buried remains were compared directly to sequences from a previous data set composed of skeletal DNA extracts from three individuals (SA, SB, and SC) who had decomposed on the ground surface (n samples = 162, 54 samples per individual). These samples were sequenced on an Illumina MiSeq platform targeting the V3-V4 region of the 16S rRNA gene using 300 PE chemistry and have previously been characterized by Emmons et al. (13 (link)) and additionally reported on by Mundorff and Davoren (39 (link)). Second, sequence data associated with samples from human feces (n = 53), sebum (n = 2), saliva (n = 6), and hair (n = 1) and associated metadata were accessed from the American Gut Project (AGP) (Qiita Accession ID 10317), a crowd-sourced project including thousands of samples from multiple body sites, with an emphasis on feces (40 (link)). The additional data set samples were selected based on donor age (50–70 years) and state of residence (TN, GA, AL, WA, and VA) to mirror the demographic information from the skeletonized individuals (Table 1; Table S3). AGP samples were processed and sequenced according to Earth Microbiome Project (EMP) protocols using 150 PE chemistry on a MiSeq Illumina Platform (Primers 515F–806R) (41 (link)).

For each sample, 16S rRNA DNA libraries were generated following a protocol described previously (47 (link)). Briefly, the V4 region of the 16S rRNA gene was targeted for PCR amplification in triplicate using 5Prime Hot master mix (QuantaBio, USA) with a modified universal bacterial 16S primer pair (515F/806R) (48 (link)). PCR products for each amplicon were pooled, run on a 2% agarose gel for visual confirmation of the libraries, and quantified with a Quant-iT PicoGreen double-stranded DNA (dsDNA) assay kit (Life Technologies, USA). One hundred nanograms of each amplicon was pooled and cleaned using a QIAquick PCR purification kit (Qiagen, USA). The library was then quantified using a Qubit dsDNA BR assay kit (Invitrogen, USA) and mixed with bacteriophage phiX DNA (10%). Paired-end sequencing (250 bp) was performed on a MiSeq platform (Illumina, USA) in iGE3, Institute of Genetics and Genomics in Geneva, CMU, University of Geneva. Sequencing results were obtained and demultiplexed using the standard method supplied by the MiSeq Illumina platform.

Forty-nine KPC-producing Enterobacterales were selected for complete sequencing, using the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA). These isolates were selected as representatives of all different hospitals, bacterial species and susceptibility profiles.
The genomic DNAs of the clinical isolates were extracted using the DNA-Sorb-B kit (Sacace Biotechnologies S.r.l., Como, Italy). Multiplexed DNA libraries were prepared using the Nextera XT library preparation kit, and 300-bp paired-end sequencing was performed on the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA) using the MiSeq v3 600-cycle reagent kit. Initial paired-end reads were quality trimmed using the Trimmomatic tool v0.33^{34 (link)} and then, assembled by use of the de Bruijn graph-based de novo assembler SPAdes v3.14.0^{35 (link)}.

PCR products were recovered using 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, United States), then were eluted by Tris–HCl, and detected by 2% agarose electrophoresis using Quantifluor-ST (Promega, United States). The products were quantified according to Illumina MiSeq platform (Illumina, San Diego, United States) Standard Operating Procedures for generating sequencing libraries from the purified amplified fragments. Finally, purified amplicons were pooled in equimolar amounts and paired-end sequenced (2 × 300 bp) on an Illumina MiSeq platform according to the standard protocols.

Amplicon libraries were prepared, as per Illumina instructions, by a 25-cycle PCR. PCR primers were the same as those used for qPCR but flanked with Illumina Nextera overhang sequences. A unique combination of Nextera XT Indices (Illumina, Cambridge, UK) were added to PCR products from each sample, via an 8-cycle PCR. PCR products were quantified using a Quant-iT™ PicoGreen™ dsDNA Assay Kit (ThermoFisher Scientific, Dartford, UK) and pooled in equimolar concentrations. Quantification of the amplicon libraries was determined via NEBNext^® Library Quant Kit for Illumina (New England BioLabs Inc., Hitchin, UK), prior to sequencing on the Illumina MiSeq^® platform (Illumina, Cambridge, UK), using a MiSeq^® 600 cycle v3 reagent kit and 20% PhiX sequencing control standard. Raw sequence data have been submitted to the European Nucleotide Archive database under accession number PRJEB37243. Sequence output from the Illumina MiSeq platform were analysed within R [50 ] using an ASV (amplicon sequence variants) bioinformatics pipeline within the DADA2 package [51 (link)]. The DADA2 pipeline included primer trimming, quality filtering, error correction and sample inference (using the DADA2 algorithm at default parameters and pooling of samples), and removal of chimeras. Finally, taxonomy assignments for ASVs were obtained using the RDP (Ribosomal Database Project) classifier [52 (link)].

NGS was conducted at KwaZulu-Natal Research Innovation and Sequencing Platform, Durban, South Africa, and Inqaba Biotechnical Industries, Pretoria, South Africa, using the Illumina MiSeq platform (Illumina, San Diego, CA). Briefly, PCR product concentrations were determined using a Qubit 3.0 fluorometer (Thermo Fisher, Malaysia). Paired-end libraries were generated using the Nextera-XT DNA library preparation kit and Nextera Index kit (Illumina, San Diego, CA), according to the manufacturer’s instructions. Sequencing libraries were purified using Agencourt AMPure XP beads, and quantified, and barcoded libraries were pooled for sequencing on an Illumina MiSeq platform. The generated raw reads (FastQ files) were assembled into contigs using online genome detection tools.^{[45 (link)]} NGS sequences were uploaded to the online variant caller polymorphism analysis sequencing (PASeq).^{[46 ]} HyDRA was also used to confirm the minor variants.^{[23 (link)]} Any variants not called by either caller were assumed to have a 0% allele frequency. Low-frequency DRMs were detected at >1% using Geneious software v8.1.9 (Biomatters Ltd, Auckland, New Zealand).^{[47 (link)]}