Supplementary materials for detection of species substitution in the meat value chain

This SUPPLEMENTARYMATERIALSreadme.txt file was generated on 2021-08-17 by JANE KAGURE NJARAMBAGENERAL INFORMATION1.

Title of Dataset: Supplementary materials for detection of species substitution in the meat value chain 

2. Author Information    

    A. Principal Investigator Contact Information
        Name: Jane Kagure Njaramba
        Institution: International Centre of Insect Physiology and Ecology (icipe)
        Address: P.O. Box 30772 - 00100, Nairobi, Kenya
        Email: njanekagure@gmail.com    

    B. Associate or Co-investigator Contact Information
        Name: Jandouwe Villinger
        Institution: International Centre of Insect Physiology and Ecology (icipe)
        Address: P.O. Box 30772 - 00100, Nairobi, Kenya
        Email: jandouwe@icipe.org
    C. Alternate Contact Information
        Name:  Lillian Wambua
        Institution: International Livestock Research Institute (ILRI)
        Address: P.O Box 30709 – 00100 Nairobi, Kenya
        Email: wambua.lillian@gmail.com

3. Date of data collection (single date, range, approximate date): 2018-11-01 to 2019-12-31

4. Geographic location of data collection: Nairobi, Kenya

5. Information about funding sources that supported the collection of the data: This study was funded by the United States
Agency for International Development Partnerships for Enhanced Engagement in Research (USAID- PEER) cycle 4, which was
awarded to Lillian Wambua and Jandouwe Villinger, under the USAID grant No. AID-OAA-A-11-00012 sub-awarded by the American
National Academy of Sciences (NAS) under agreement No. 2000006204. Further support was provided by icipe institutional
funding from the UK’s Foreign Commonwealth and Development Office (FCDO), the Swiss Agency for Development and Cooperation
(SDC), the Swedish International Development Cooperation Agency (SIDA), and the Kenyan government. These funding bodies did
not play a role in the design of this study, the data collection, interpretation of the data, or writing and submission of
this publication.
 
ABSTRACT:This data set is linked to the study that displays the use of PCR-HRM to distinguish between different meat sources
by targeting the mitochindrial markers Cytochrome c Oxidase subunit 1 (CO1), Cytochrome b (Cyt b) and the 16s subunit of
ribosomal RNA (16S rRNA). The combined use of the three markers allowed for the distinction of the species. Additionally,
the utility of PCR-HRM was shown in raw, cooked, dried and rotten, as well as pure and mixed meat samples.

Key words:species substitution, meat value chain, high-resolution melting analysis, 16S rRNA, cytochrome c oxidase subunit
1, cytochrome b

SHARING/ACCESS INFORMATION

1. Licenses/restrictions placed on the data:  Open Access
2. Links to publications that cite or use the data: None
3. Links to other publicly accessible locations of the data: https://doi.org/10.1101/2021.01.12.426171 
4. Links/relationships to ancillary data sets:None 
5. Was data derived from another source? yes/no:  No
    A. If yes, list source(s): 
6. Recommended citation for this dataset: Njaramba Jane Kagure, Lillian Wambua, Titus Mukiama, Nelson Onzere Amugune, and
Jandouwe Villinger(2021). Supplementary Materials

DATA & FILE OVERVIEW
1. File List: <list all files (or folders, as appropriate for dataset organization) contained in the dataset, with a brief
description>
Folder Name: Supplementary Information 

a) Supplementary Table 1- Metadata of voucher specimen 
Description:Metadata of voucher specimen used as positive controls in this study.  Meat samples from known vertebrate
species selected for use as positive controls in the PCR-HRM analyses. 

b)Supplementary Figure 1- PCR-HRM profiles of reference livestock species
Title:Supplementary Figure 1:  Distinct normalized PCR-HRM profiles of meat from seven reference livestock species. 
Figure Legend: HRM profiles are represented as percent fluorescence with increase in temperature for (a) COI, (b) cyt b, and
(c) 16S rRNA markers. All unknown samples were compared against these reference profiles for identification of vertebrate
species. 

c)Supplementary Figure 2- Box plots of meat samples exposed to different physicochemical conditions
Title: Supplementary Figure 2: Box plots of peak melting temperatures (°C) of meat samples exposed to different
physicochemical conditions.  
Figure Legend: Replicate meat samples from goat, sheep, pig, cattle, camel, and chicken were exposed to different
conditions; raw, rotten, oven-dried, and microwaved. The peak PCR-HRM melt rate temperatures were plotted for a) CO1, b) cyt
b, and c) 16S rRNA markers.

d)Supplementary Figure 3- PCR-HRM distinction of doubled peaked profiles 
Title: Supplementary Figure 3: PCR-HRM profiles showing the distinction of species presenting with double peaks. Figure
Legend: Unique double peaks produced by samples from cattle, sheep, pig, chicken and camel samples when targeting cyt b and
CO1 allowed for easier identification of these vertebrates. This differentiation relied on the melt profiles, through the
analysis of multiple peaks produced in the amplification of a) CO1 and b) cyt b.

e)Supplementary Figure 4- Box plots of meat samples with DNA extracted using different protocols
Title: Supplementary Figure 4: Box plots of peak melting temperature (°C) seen in meat samples extracted using different
extraction protocols. 
Figure legend: DNA from four vertebrate species (two cattle, four goats, one sheep and two camels) were extracted in
replicate using two commercial kits; DNeasy Blood and Tissue Kit protocol, the ISOLATE II Genomic DNA Extraction Kit, and
two manual extraction protocols. The peak PCR-HRM melt rate temperatures were plotted for a) CO1, b) cyt b, and c) 16S rRNA
markers. 
2. Relationship between files, if important: N/A

3. Additional related data collected that was not included in the current data package: None
4. Are there multiple versions of the dataset? yes/no:  No
    A. If yes, name of file(s) that was updated:         
        i. Why was the file updated?
         ii. When was the file updated?

 METHODOLOGICAL INFORMATION

1.Description of methods used for collection/generation of data: <Include links or references to publications or other
documentation containing experimental design or protocols used in data collection>
Meat samples were purchased from randomly selected meat stalls in  in Nairobi’s major meat wholesale market (Burma market),
and butcheries from the surrounding residential areas.DNA was extracted from the meat samples using different extraction
protocols and vertebrate meat species identified by amplification of the  mitochondrial markers CO1, cyt b and 16S rRNA. The
distinction was possible through the use of high-resolution melting analysis of the Polymerase Chain Reaction (PCR)
products. Theprotocols used are similar to those described by Ouso et al., 2020. 

2. Methods for processing the data: <describe how the submitted data were generated from the raw or collected data>
Representative PCR products were sent to Macrogen Inc. (Netherlands) for Sanger sequencing. The sequences were then edited,
trimmed and queried against the NCBI database using BLAST parameters. Sequences were analyzed using Geneious version 11.1.5. 

3. Instrument- or software-specific information needed to interpret the data: <include full name and version of software,
and any necessary packages or libraries needed to run scripts>
The RotorGene Q thermocycler (Qiagen, Germany) was used to carry out the PCR coupled with high-resolution melting. It's
accompanying software was used for the analysis of the melt curves. The Box-plots were generated using the statistical
software NCSS 2020 (NCSS, Kaysville, Utah, USA).

4. Standards and calibration information, if appropriate: Twenty-four reference meat samples of known vertebrates archived
from a previous study (Ouso et al., 2020) were used as positive controls.

5. Environmental/experimental conditions: To study the effect of various physicochemical conditions of meat samples on
species identification by PCR-HRM analysis, we utilized sub-samples from our collection of positive controls. They were
exposed to different treatments to simulate fresh, dried, cooked (microwaved), and rotting/decomposed meat.

6. Describe any quality-assurance procedures performed on the data: Experiments were carried out in replicates to ensure
that the data was reliable. Additionally, negative controls, where applicable, were used.  

7. People involved with sample collection, processing, analysis and/or submission: Jane Njaramba, Lillian Wambua, Jandouwe
Villinger

DATA-SPECIFIC INFORMATION FOR: Supplementary Table 1- Metadata of voucher specimen 

1. Number of variables: 5
2. Number of cases/rows: 8 

3. Variable List: 
a)Scientific name- gives the binomial name of the species used as reference samples. 
b)Origin of sample- describes where the reference sample was obtained
c)GPS Address- Provides the Geolocation of where the sample was collected from 
d)Reference- A citation of previous literature where the same samples were used as references
e)No. of replicates- Shows the number of replicates used in the study 

4. Missing data codes:
'-' indicates that there was no reference for the samples as they had not been used in previous work

5. Specialized formats or other abbreviations used: 'No.' refers to 'Number' 

DATA-SPECIFIC INFORMATION FOR: Supplementary Figure 1- PCR-HRM profiles of reference livestock species

1. Number of variables: 3
2. Number of cases/rows: 8

3. Variable List:
a)Normalized Fluorescence (%)- This shows the change in fluoresence with increase in temperature
b)Temperature- Indicates the change in temperature in degrees celsius
c)Mitochondrial markers- Each marker is (CO1, Cyt b and 16S rRNA) have distinct melt curves  

4. Missing data codes: None

5. Specialized formats or other abbreviations used: None

DATA-SPECIFIC INFORMATION FOR: Supplementary Figure 2- Box plots of meat samples exposed to different physicochemical
conditions
1. Number of variables: 3
2. Number of cases/rows: 6

3. Variable List:
a)Treatment- This refers to the physicochemical conditions the samples were in. The treatments were applied to simulate
fresh, dried, cooked (microwaved), and rotting/decomposed meat.
b)Melting Temperature- Indicates the melting temperature for the replicate samples per species in degree celsius
c)Mitochondrial markers- The melting temperature for each marker(CO1, Cyt b and 16S rRNA) in selected vertebrate species.  

4. Missing data codes: None

5. Specialized formats or other abbreviations used: None

DATA-SPECIFIC INFORMATION FOR:Supplementary Figure 3- PCR-HRM distinction of doubled peaked profiles 
1. Number of variables: 3
2. Number of cases/rows:21 

3. Variable List:
a)Melt rate (dF/dT)- This shows the rate at which the double stranded-DNA melts, resulting in a reduction in fluorescence.
It is measure as the change in fluorescence (dF) divided by the change in temeprature (dT).
b)Temperature- his shows the change in temperature. Measured in degrees celsius.
c)Mitochondrial markers- The mitochondrial markers CO1 and Cyt b were displayed as these markers have double peaks in
specific species.
4. Missing data codes:None

5. Specialized formats or other abbreviations used: None

DATA-SPECIFIC INFORMATION FOR: Supplementary Figure 4- Box plots of meat samples with DNA extracted using different
protocols
1. Number of variables: 3
2. Number of cases/rows: 4

3. Variable List: <list variable name(s), description(s), unit(s)and value labels as appropriate for each>
a)Protocol- This refers to the different DNA extraction protocols used on the samples. These included the use of two
commercial kits ISOLATE II Genomic DNA Kit, and the DNeasy Blood and Tissue Kit protocol and a lab-optimized  and modified
lab extraction protocol.
b)Melting Temperature- Indicates the melting temperature for the replicate samples per species in degree celsius
c)Mitochondrial markers- The melting temperature for each marker(CO1, Cyt b and 16S rRNA) in selected vertebrate species.  

4. Missing data codes:None

5. Specialized formats or other abbreviations used: None