Additional file 1 of Variation in gut bacterial composition is associated with Haemonchus contortus parasite infection of sheep
Creators: name: Md. Mamun
name: Sandeman, Mark
name: Rayment, Phil
name: Brook-Carter, Phillip
name: Scholes, Emily
name: Naga Kasinadhuni
name: Piedrafita, David
name: Greenhill, Andrew
Publisher: name: figshare
Year: 2020
Subjects: subject: Medicine
subject: Ecology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: 69999 Biological Sciences not elsewhere classified
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: 60506 Virology

description: Additional file 1. ARISA results. Community Structure of Faecal Microbiota of High- and Low-Burden Sheep Prior to Infection.A three-way ANOSIM of ARISA data revealed a separation (R = 0.243, p ≤ 0.01) of microbial composition between high- and low-burden sheep. Differences in the microbial composition were visualised by nMDS plot, showing distinct clustering of faecal microbiota in high-burden sheep relative to low-burden sheep (Additional file 1: Figure S10). Most OTUs were shared among high and low parasite burden sheep groups (Additional file 1: Figure S11), but abundances of dominant OTUs differed (Additional file 1: Figure S12). Community Structure of Faecal Microbiota of High- and Low-Burden Sheep Before and After H. contortus Infection. Three-way ANOSIM of ARISA data revealed separation (R = 0.243, p ≤ 0.01) of gut microbial composition following H. contortus infection in high-burden sheep, and no clear separation in low-burden sheep (R = 0.190, p ≤ 0.01). An nMDS plot showed a lack of distinct clustering of gut microbiota in uninfected and infected sheep with high worm burden, although there was a predominance of infected sheep samples in the upper left section of the plot (Additional file 1: Figure S13a). No clear clustering was observed in low-burden sheep (Additional file 1: Figure S13b). There were no significant differences in the relative abundances of the dominant OTUs following infection (Figure S14). ARISA data analysis. ARISA was used to determine community profiles. Quantified DNA was diluted to 20 ng/μL using RNase free water for use as template for PCR [63, 64]. PCR amplification of the ITS region was performed in duplicate using the previously described primer set ITSF/ITSReub [65–68] with HotStarTaq® Plus master mix. Fragment separation was conducted using an Applied Biosystems 3730 DNA analyser with a GS1200 LIZ® internal size standard. Peak size, height and area data were extracted to Microsoft Excel after performing accurate size calling by using GeneMapper software Version 4.0 for further analysis. The software converted fluorescence data into electropherograms; peaks represented fragments of different sizes, and the peak’s areas represented the relative proportion of the fragments. All peaks with fluorescent intensity of ≤50 relative fluorescence units were excluded as they might be the part of instrumental noise (sometimes referred to as background peaks) [64, 66, 69–71]. Given the approximate minimal known lengths of the ITS region (143 bp) [70] included in the primer sets ITSC and 1552/132, fragment lengths below 229 bp and 300 bp, respectively, were eliminated from analysis. Data comprising the true peak sizes and peak areas were converted to abundance per binned operational taxonomic units (OTUs) using the custom binning script interactive binner [64] in the R software package [72], with a relative fluorescence intensity cut-off of 0.09%, a window size (WS) of two and a shift size of 0.1 [64]. To determine the best binning strategy for a dataset without a priori knowing the ideal WS value, the script automatic binner [64] in R was used which allows for an automatic calculation of a series of WS values (e.g. 0.5, 1, 2, 3, 4, and 5 bp) for a given shift value (e.g. 0.1 bp). A compromise between high resolution (low WS) and high similarity among samples (high WS) was made based on the output of the script. Figure S1 Rarefaction curve based on OTU richness values. Figure S2 The bacterial families with significant differences in relative abundances in high and low burden sheep (total family = 133). The Y-axis shows the average relative abundances (%); X-axis shows family. Unpaired t-test. Error bars represent SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Figure S3 Venn diagram representing the shared and unique OTUs in sheep with high and low burdens of parasite. A bacterial group was considered to be present in a sample group if it was identified in at least 10% of the samples within the group. Figure S4 Venn diagram representing the core OTUs in group of sheep with high and low burden of parasite. A bacterial group was considered to be present in a sample group if it was identified in at least 50% of the samples within the group. Figure S5 HeatMap + of the relative abundances of the identified phyla in uninfected and infected sheep. (a) high-burden; (b) low-burden sheep. The maps showed marked differences in relative abundances in high-burden sheep compared to low-burden sheep. Figure S6 The bacterial families with significant differences in relative abundances before and after infection. (a) high-burden; (b) low-burden sheep. The Y-axis shows the average relative abundances (%); X-axis shows family. Pair-wise comparisons are done by unpaired t-test. Error bars represent SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Figure S7 Venn diagram representing the shared and unique OTUs present in sheep before and after infection. (a) high-burden sheep; (b) low-burden sheep. A bacterial group was marked as present in a sample group if it was identified in at least 10% of the samples within the group. Figure S8 Venn diagram representing the core OTUs present in sheep before and after infection. (a) high-burden sheep; (b) low-burden sheep. An OTU was considered as core if it was identified in at least 50% of the samples within the group. Figure S9 The two most dominant bacterial phyla with significant differences in relative abundances between high- and low burden sheep prior to infection. The Y-axis shows the average relative abundances (%); X-axis shows phyla. Significance determined using Mann Whitney test. Error bars represent SD. ****p ≤ 0.0001. Figure S10 Distinct clustering of gut microbiome between high and low parasite burden sheep (before infection) using ARISA data. nMDS plot based on Bray-Curtis similarity matrix of gut bacterial composition (10 sheep, 4 sampling events). Each symbol represents an individual sheep sample at a given time. Blue indicates high-burden and red represents low-burden sheep. Figure S11 Venn diagram representing the shared and unique OTUs in high and low parasite burden sheep as determined by ARISA sampled over 4 weeks. An OTU was considered to be present in a sample group if it was identified in at least one of the samples within the group. Figure S12 Average relative abundances of 10 most dominant OTUs of high-burden and low-burden sheep, as determined by ARISA. The Y-axis shows the average relative abundances (%). X-axis represents OTUs. Unpaired t- test. Error bars represent SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Figure S13 Clustering of gut microbiome of uninfected and infected sheep using ARISA data. (a) high-burden; (b) low-burden sheep. nMDS plot of sheep based on Bray-Curtis similarity matrix of faecal microbial composition. Blue indicates uninfected whereas red represents infected sheep sample. Sampling were conducted over eight weeks, giving rise to 40 dot points for both high and low burden sheep. Figure S14 Average relative abundances of commonly detected OTUs of uninfected and infected sheep detected by ARISA. (a) high-burden; (b) low-burden sheep. The Y-axis shows the average relative abundances (%). X-axis represents OTUs. Error bars represent SD. (Paired t-test; no significance was observed). Table S1 ANOSIM of microbial composition of sheep between high-burden and low-burden sheep (16S data). **p ≤ 0.01. Table S2 Identified genera with significantly different abundances between the high and low burden sheep (16S data). Significance determined using the Wilcoxon matched pairs signed rank test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Table S3 The 50 most abundant core OTUs with different abundances between the high and low parasite burden sheep (16S data). Table S4 ANOSIM of microbial composition of high and low parasite burden sheep following infection (16S data). **p ≤ 0.01, ***p ≤ 0.001. Table S5 The genera with significantly different abundances between the uninfected and infected sheep with high parasite burden (n = 20) (16S data). Significance determined using the Wilcoxon matched pairs signed rank test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Table S6 The genera with significantly different abundances between the uninfected and infected sheep with low parasite burden (n = 20) (16S data). Significance determined using the Wilcoxon matched pairs signed rank test. *p ≤ 0.05, **p ≤ 0.01