To obtain a comprehensive picture of microbial communities within a sample, it is essential to choose a sampling method which absorbs and preserves biological materials during sampling, transport and storage until DNA extraction. The effectiveness of a swab may be influenced by a number of factors, most importantly the material of the swab tip which can affect the rate at which bacteria are collected during the sampling process. Furthermore, the design of the transport tube as well as the DNA preserving liquids can affect the integrity of the material during transport. Finally, the amount of background contamination identified for different products should be taken into account.

Surface samples were collected and preserved using a flocked swab with a storage tube containing a buffer that is optimized for DNA preservation. Two different sets of materials were used for collection in 2016 and 2017.

In the first method of sample collection used a Copan Liquid Amies Elution Swab (ESwab, Copan Diagnostics, Cat.: 480C) paired with a 1mL of Liquid Amies in a plastic, screw cap tube, hereafter referred to as a ‘Copan swab’. The Amies transport medium maintains the sample at pH 7.0 0.5 and contains sodium thioglycolate as well as calcium, magnesium, sodium, and potassium salts to control the permeability of bacterial cells. Once the surface was sampled, the swab was immediately placed into the collection tube and stored in a −80C freezer once returned to the laboratory.

The second method used an individually wrapped Isohelix Buccal Mini Swab (MS Mini DNA/RNA Swab, Isohelix, Cat.: MS-02) paired with a barcoded storage tubes (2D Matrix V-Bottom ScrewTop Tubes, Thermo Scientific, Cat.: 3741-WP1D-BR/1.0mL), hereafter referred to as ‘matrix tubes’, prefilled with 400μl of a transport and storage medium suitable for both DNA and RNA (DNA/RNA Shield, Zymo Research, Cat.: R1100), hereafter referred to as ’Zymo Shield’. Once the surface was sampled, the swab was immediately placed into a matrix tube containing Zymo Shield and stored in a −80C freezer until DNA extraction.

We assessed the absorption strength of both the Copan and Isohelix swabs for various biological and surface materials encountered when sampling metro stations. A single surface was selected for a designated sampling area to test the absorption strength. Both swabs were moistened by submerging the swab for a few seconds in their preservative media. The area was then swabbed for 3 min, covering the selected surface. By moistening the swab prior to sampling, the swab matrix would take up more microflora already saturated with the transport medium.

A standard operating procedure (SOP) was developed for the sample collection to be followed by all members of the MetaSUB consortium participating in CSD, and adapted from earlier work by Afshinnekoo et al. (2015). The aim was to standardize as much of the sampling procedure in order to ensure high quality control across the various cities and sampling teams. Thus, it was recommended that teams collect samples from high contact surfaces found in most metro and transit stations and systems around the world, including ticket kiosks, turnstiles, railings, and seats or benches. Some cities had to adapt the sampling approach to better reflect their city. For example, in cases where a city did not have a subway system, the most common form of public transit was studied instead. While variation in the types of surfaces being sampled were allowed, modifications to the sampling procedure itself were not. Moreover, a number of metadata were recorded for each sample during the process of collection to ensure as much contextual information as possible was captured. Each city developed their own sampling and submitted them for review before sampling kits were sent to them in order to ensure consistency across the various sites.

All principal investigators and MetaSUB city leaders were trained in the sampling protocol and this training was further disseminated to the respective sampling teams to ensure consistent and quality control sampling. Each participant was instructed to don disposable latex or nitrile gloves prior to sample collection. The swab was dipped in the preservative medium for approximately 2 s before the swab was firmly dragged across the surface, using both sides and using different angles, for a total of 3 min to ensure highest yield. Any other important notes or observations could be added to the metadata for each sample.

A sampling protocol video overview is included in the Supplemental information.

To assess the quality of our sampling procedure, we created multiple controlled scenarios. As a positive laboratory control, a Copan swab was introduced into a sterile urine cup with 30μl of a well-defined, accurately characterized microbial reference sample (ZymoBIOMICS Microbial Community Standard, Zymo Research, Cat.: D6300). A negative control was made by adding 50μl of the final resuspension buffer from the DNA isolation step into a sterile urine cup before introducing a Copan swab. Furthermore, a laboratory workbench was swabbed using our sampling procedure both before and after it was cleaned with a 10% bleach solution. To detect background contamination due to biological material in the air in sample areas, a dampened Copan swab was held in the air for approximately 3 min. Finally, to ensure there was no contamination could be due to the consumables we procured prior to sampling, we also swabbed, in triplicate, the interior of a flow hood that had been sterilized with 10% bleach before wiping down with ethanol and irradiating with ultraviolet light.

Metadata from individual cities was collected from a standardized form and set of data fields. The principal fields collected were the location of sampling, the material of the object being sampled, the type of object being sampled, the elevation above or below sea level, and the station or line where the sample was collected. However, several cities were unable to use the provided software application for various reasons, and instead submitted their metadata as separate spreadsheets that could be added to the data repository. Additionally, certain metadata features, such as those related to sequencing and quality control, were added after initial sample collection. To collate various metadata sources, we built a publicly available repository on Pangea (https://pngb.io/metasub-2021) which assembled a large master spreadsheet with consistent sample universally unique identifiers (UUID). After assembling the originally collected data attributes we added normalized attributes based on the original metadata to account for surface material, control status, and features of individual cities. A full description of ontologies used is provided as part of the collating program.

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