scholarly journals Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2093
Author(s):  
Shen-Yuan Hsieh ◽  
Mohammad A. Tariq ◽  
Andrea Telatin ◽  
Rebecca Ansorge ◽  
Evelien M. Adriaenssens ◽  
...  

The human intestinal microbiota is abundant in viruses, comprising mainly bacteriophages, occasionally outnumbering bacteria 10:1 and is termed the virome. Due to their high genetic diversity and the lack of suitable tools and reference databases, the virome remains poorly characterised and is often referred to as “viral dark matter”. However, the choice of sequencing platforms, read lengths and library preparation make study design challenging with respect to the virome. Here we have compared the use of PCR and PCR-free methods for sequence-library construction on the Illumina sequencing platform for characterising the human faecal virome. Viral DNA was extracted from faecal samples of three healthy donors and sequenced. Our analysis shows that most variation was reflecting the individually specific faecal virome. However, we observed differences between PCR and PCR-free library preparation that affected the recovery of low-abundance viral genomes. Using three faecal samples in this study, the PCR library preparation samples led to a loss of lower-abundance vOTUs evident in their PCR-free pairs (vOTUs 128, 6202 and 8364) and decreased the alpha-diversity indices (Chao1 p-value = 0.045 and Simpson p-value = 0.044). Thus, differences between PCR and PCR-free methods are important to consider when investigating “rare” members of the gut virome, with these biases likely negligible when investigating moderately and highly abundant viruses.

2019 ◽  
Author(s):  
Rachel L. Marine ◽  
Laura C. Magaña ◽  
Christina J. Castro ◽  
Kun Zhao ◽  
Anna M. Montmayeur ◽  
...  

ABSTRACTNext-generation sequencing is a powerful tool for virological surveillance. While Illumina® and Ion Torrent® sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. We evaluated the Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of assembled viral genomes, and an estimation of cost per sample to generate the data was calculated. The choice of library preparation kit and sequencing platform was found to impact the breadth of genome coverage and accuracy of consensus viral genomes. The Ion Torrent S5 outperformed the older Ion Torrent PGM platform in data quality and cost, and generated the highest proportion of reads for enterovirus D68 samples. However, indels at homopolymer regions impacted the accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., Ion Torrent 510 or Illumina MiSeq Nano V2), the cost per sample was lower on the MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 or Illumina MiSeq V2) the cost per sample was comparable. These findings suggest that the Ion Torrent S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of RNA viruses, each with specific advantages and tradeoffs.


2020 ◽  
Vol 38 (6_suppl) ◽  
pp. 730-730 ◽  
Author(s):  
Anika Agarwal ◽  
Jennifer Modliszewski ◽  
Lauren Davey ◽  
Marco Reyes-Martinez ◽  
Daniella Runyambo ◽  
...  

730 Background: ICIs are effective in mRCC, but one pertinent clinical need is to identify predictive biomarkers for response. The PD-1 receptor has been implicated in regulating gastrointestinal commensal bacteria, with varied immune interactions, thereby impacting response to ICIs. We evaluated bacterial taxa and ICI outcomes in mRCC pts. Methods: Fecal samples from 22 mRCC pts were collected at baseline, week (wk)-4 on ICI, and upon disease progression. Pts were grouped as responders (R, complete or partial response) or non-responders (NR, stable or progressive disease). Microbial DNA was isolated by next generation DNA sequencing. The V4 region of bacterial 16S ribosomal RNA was amplified from extracted DNA and analyzed for bacterial abundance, as well as alpha diversity indices (number of amplicon sequence variants [ASVs], Shannon’s Index, Faith’s Phylogenetic Diversity, and Pielou’s evenness) and beta diversity indices on ASVs (Bray-Curtis, Jaccard, and unweighted/weighted UniFrac dissimilarity measures). Results: Beta diversity analysis at baseline showed no difference in microbial composition between Rs and NRs. However, beta diversity analysis did show a significant change in composition from baseline to wk 4 in R vs NR pts (Bray Curtis p-value=0.03). Among mRCC pts with CR to ICIs, counts of bacteria in the phylum Verrucomicrobia had an upward trend from baseline to wk 4. All mRCC pts with CR (n=3) had Akkermansia at wk 4. However, Akkermansia colonization was not sufficient for response, present in 7/9 Rs and 6/11 NRs. Conclusions: Baseline microbiome differences between ICI Rs and NRs are not enough to predict outcomes. Diversity changes between baseline and wk-4 on treatment could be an early predictor of response. Factors other than presence of Akkermansia (tumor or host-specific, Akkermansia strain variation, or other bacteria in the microenvironment) may contribute to response. Further species and strain-level profiling of the microbiota, tumor-specific genomic alterations, host immune response, and increasing sample size of ICI-treated patients may improve detection of significant differences between Rs and NRs.


2021 ◽  
Author(s):  
Amrapali Rajput ◽  
Shipeng Zhou ◽  
Madhava Meegaskumbura

It is known that animal-associated microbiomes form indispensable relationships with hosts and are responsible for many functions important for host-survival. Next-gen driven approaches documenting the remarkable diversity of microbiomes have burgeoned, with amphibians too, benefiting from such treatments. The microbiome of Gymnophiona (caecilians), one of the three amphibian orders, constituting of 3% of amphibians, however, remains almost unknown. The present study aims to address this knowledge gap through analysis of the microbiome of Ichthyophis bannanicus. As these caecilian larvae are aquatic and hence exposed to a greater propensity for bacterial microbiomic interchange, we hypothesize that bacterial phyla would overlap between gut and skin. Further, from the host-specificity patterns observed in other vertebrate taxa, we hypothesize that Gymnophiona have different dominant gut bacterial microbiomes at a higher taxonomic level when compared to the larvae of the other two amphibian orders (Anura and Caudata). We used 16S rRNA gene amplicon sequencing based on Illumina Nova sequencing platform to characterize and compare the gut (represented by faecal samples) and skin microbiome of I. bannanicus larvae (N = 13), a species distributed across South-East-Asia and the only caecilian species occurring in China. We compared our gut microbiome results with published anuran and caudate larval microbiomes. For I. bannanicus, a total of 4,053 operational taxonomic units (OTU) across 13 samples were detected. Alpha-diversity indices were significant between gut and skin samples. Non-metric multidimensional scaling analysis suggest that gut and skin samples each contained a distinct microbiome at OTU level. We record significant differences between the bacterial phyla of gut and skin samples in larvae of I. bannanicus. The study provides an overview of gut and skin bacterial microbiomes of a caecilian, while highlighting the major differences between larval microbiomes of the three amphibian orders. We find a partial overlap of gut bacterial microbiomes at phylum level for the three orders; however, the relative abundance of the dominant phyla is distinct. The skin and gut microbiomes are distinct with little overlap of species, highlighting that gut-skin axis is weak. This in turn suggests that many of the microbial species on skin and gut are functionally specialized to those locations. We also show that the skin microbiome is more diverse than the gut microbiome at species level; however, a reason for this could be a portion of the gut microbiome not being represented in faecal samples. These first microbiome information from a caecilian lay the foundation for comparative studies of the three amphibian orders.


2020 ◽  
Vol 2020 ◽  
pp. 1-8 ◽  
Author(s):  
Cheng-Xing Long ◽  
Hao-Qing Shao ◽  
Cheng-Yu Luo ◽  
Rong Yu ◽  
Zhou-Jin Tan

The current research tried to explore the effect of Qiweibaizhu powder (QWBZP) on the bacterial diversity and community structure of the intestinal mucosa of dysbiosis diarrhea mice and provide a scientific basis for the efficacy of QWBZP on antibiotic-induced diarrhea. A dysbiosis diarrhea mouse model was constructed with broad-spectrum antibiotics through a mixture of cephradine capsules and gentamicin sulfate (23.33 mL·kg-1·d-1). Intestinal mucosa was collected, and DNA was extracted from each group. The bacterial characteristics in intestinal mucosa were analyzed by MiSeq sequencing based on the 16S rRNA sequencing platform. There were no significant differences in alpha diversity indices among the three groups. The sample distributions in both the normal and QWBZP groups were relatively concentrated, and the distance among individuals was close. However, an opposite result was obtained in the model group. Furthermore, the composition and abundance of species were similar between the normal group and the QWBZP group at both the phylum and genus levels. After treatment with QWBZP, the abundance of Lactobacillus increased, and Proteobacteria decreased, and the Firmicutes/Bacteroidetes ratio decreased to a normal level. Our results indicate that QWBZP can help repair mucosal bacterial structure and recover mucosal microbiota. Specifically, QWBZP increased the abundance of Lactobacillus and Bacteroidales S24-7 group norank.


2021 ◽  
Author(s):  
Yangyang JIA ◽  
Shengguo Zhao ◽  
Wenjie Guo ◽  
Ling Peng ◽  
Fang Zhao ◽  
...  

Abstract BackgroundDue to the inherent scarcity of the microbial rare taxa, it is difficult to distinguish between bona fide rare taxa and potential false positives in metabarcoding amplicon sequencing studies. Although recently developed quality control and clustering or denoising algorithms could remove sequencing errors or non-biological artifact reads, no algorithm could remove high quality reads from sample-wise cross contaminations introduced by index misassignment. ResultsWe thoroughly evaluated the rate of index misassignment of the mostly used NovaSeq 6000 and DNBSEQ-G400 sequencing platforms using both commercial and customized mock communities, and observed significant higher (5.68% vs 0.08%) fraction of potential false positive reads for NovaSeq 6000 compared to DNBSEQ-G400 in. Significant batch effects could be caused by the randomly detected false positive or false negative rare taxa. These false detections introduced by index misassignment could also lead to inflated alpha diversity for relatively simple samples while underestimated alpha diversity for complex samples. Further test using a set of cow rumen samples reported differential rare taxa by different sequencing platforms. Correlation test of the rare taxa detected by each sequencing platform demonstrated that the Novaseq 6000 platform detected rare taxa had a much lower possibility to be correlated with the physiochemical properties of rumen fluid. Community assembly mechanism and microbial network association analysis indicated that false positive or negative rare taxa detection could lead to distorted community assembly process and identification of even fake keystone species of the community, one of which was confirmed negative by PCR cloning and following Sanger sequencing. ConclusionsMetabarcoding amplicon sequencing process may introduce scarce but not neglectable false positives. We highly suggest proper positive and negative controls and technical replicate settings, and proper sequencing platform selection in future amplicon studies, especially when the microbial rare biosphere should be focused.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hua Zha ◽  
Fengping Liu ◽  
Zongxin Ling ◽  
Kevin Chang ◽  
Jiezuan Yang ◽  
...  

AbstractType 2 diabetes mellitus (T2DM) influences the human health and can cause significant illnesses. The genitourinary microbiome profiles in the T2DM patients remain poorly understood. In the current study, a series of bioinformatic and statistical analyses were carried out to determine the multiple bacteria associated with the more dysbiotic genitourinary microbiomes (i.e., those with lower dysbiosis ratio) in T2DM patients, which were sequenced by Illumina-based 16S rRNA gene amplicon sequencing. All the genitourinary microbiomes from 70 patients with T2DM were clustered into three clusters of microbiome profiles, i.e., Cluster_1_T2DM, Cluster_2_T2DM and Cluster_3_T2DM, with Cluster_3_T2DM at the most dysbiotic genitourinary microbial status. The three clustered T2DM microbiomes were determined with different levels of alpha diversity indices, and driven by distinct urinalysis variables. OTU12_Clostridiales and OTU28_Oscillospira were likely to drive the T2DM microbiomes to more dysbiotic status, while OTU34_Finegoldia could play a vital role in maintaining the least dysbiotic T2DM microbiome (i.e., Cluster_1_T2DM). The functional metabolites K08300_ribonuclease E, K01223_6-phospho-beta-glucosidase and K00029_malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+) were most associated with Cluster_1_T2DM, Cluster_2_T2DM and Cluster_3_T2DM, respectively. The characteristics and multiple bacteria associated with the more dysbiotic genitourinary microbiomes in T2DM patients may help with the better diagnosis and management of genitourinary dysbiosis in T2DM patients.


2021 ◽  
Vol 11 (1) ◽  
pp. 35
Author(s):  
Zahra A. Barandouzi ◽  
Joochul Lee ◽  
Kendra Maas ◽  
Angela R. Starkweather ◽  
Xiaomei S. Cong

The interplay between diet and gut microbiota has gained interest as a potential contributor in pathophysiology of irritable bowel syndrome (IBS). The purpose of this study was to compare food components and gut microbiota patterns between IBS patients and healthy controls (HC) as well as to explore the associations of food components and microbiota profiles. A cross-sectional study was conducted with 80 young adults with IBS and 21 HC recruited. The food frequency questionnaire was used to measure food components. Fecal samples were collected and profiled by 16S rRNA Illumina sequencing. Food components were similar in both IBS and HC groups, except in caffeine consumption. Higher alpha diversity indices and altered gut microbiota were observed in IBS compared to the HC. A negative correlation existed between total observed species and caffeine intake in the HC, and a positive correlation between alpha diversity indices and dietary fiber in the IBS group. Higher alpha diversity and gut microbiota alteration were found in IBS people who consumed caffeine more than 400 mg/d. Moreover, high microbial diversity and alteration of gut microbiota composition in IBS people with high caffeine consumption may be a clue toward the effects of caffeine on the gut microbiome pattern, which warrants further study.


2021 ◽  
Vol 22 (4) ◽  
pp. 2131
Author(s):  
Stefania Pane ◽  
Anna Sacco ◽  
Andrea Iorio ◽  
Lorenza Romani ◽  
Lorenza Putignani

Background: Strongyloidiasis is a neglected tropical disease caused by the intestinal nematode Strongyloides stercoralis and characterized by gastrointestinal and pulmonary involvement. We report a pediatric case of strongyloidiasis to underline the response of the host microbiota to the perturbation induced by the nematode. Methods: We performed a 16S rRNA-metagenomic analysis of the gut microbiota of a 7-year-old female during and after S. stercolaris infection, investigating three time-point of stool samples’ ecology: T0- during parasite infection, T1- a month after parasite infection, and T2- two months after parasite infection. Targeted-metagenomics were used to investigate ecology and to predict the functional pathways of the gut microbiota. Results: an increase in the alpha-diversity indices in T0-T1 samples was observed compared to T2 and healthy controls (CTRLs). Beta-diversity analysis showed a shift in the relative abundance of specific gut bacterial species from T0 to T2 samples. Moreover, the functional prediction of the targeted-metagenomics profiles suggested an enrichment of microbial glycan and carbohydrate metabolisms in the T0 sample compared with CTRLs. Conclusions: The herein report reinforces the literature suggestion of a putative direct or immune-mediated ability of S. stercolaris to promote the increase in bacterial diversity.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Calvin P. Sjaarda ◽  
Nazneen Rustom ◽  
Gerald A. Evans ◽  
David Huang ◽  
Santiago Perez-Patrigeon ◽  
...  

AbstractThe emergence and rapid global spread of SARS-CoV-2 demonstrates the importance of infectious disease surveillance, particularly during the early stages. Viral genomes can provide key insights into transmission chains and pathogenicity. Nasopharyngeal swabs were obtained from thirty-two of the first SARS-CoV-2 positive cases (March 18–30) in Kingston Ontario, Canada. Viral genomes were sequenced using Ion Torrent (n = 24) and MinION (n = 27) sequencing platforms. SARS-CoV-2 genomes carried forty-six polymorphic sites including two missense and three synonymous variants in the spike protein gene. The D614G point mutation was the predominate viral strain in our cohort (92.6%). A heterozygous variant (C9994A) was detected by both sequencing platforms but filtered by the ARTIC network bioinformatic pipeline suggesting that heterozygous variants may be underreported in the SARS-CoV-2 literature. Phylogenetic analysis with 87,738 genomes in the GISAID database identified global origins and transmission events including multiple, international introductions as well as community spread. Reported travel history validated viral introduction and transmission inferred by phylogenetic analysis. Molecular epidemiology and evolutionary phylogenetics may complement contact tracing and help reconstruct transmission chains of emerging diseases. Earlier detection and screening in this way could improve the effectiveness of regional public health interventions to limit future pandemics.


2009 ◽  
Vol 99 (1) ◽  
pp. 5-11 ◽  
Author(s):  
Laura I. Weber ◽  
Cintia G. Hildebrand ◽  
Anderson Ferreira ◽  
Gustavo Pedarassi ◽  
José A. Levy ◽  
...  

A genetic study of the neotropical river otter Lontra longicaudis (Olfers, 1818), which has an unknown conservation status, was carried out at the Taim Ecological Station and the margins of the Vargas stream, Rio Grande do Sul, southern Brazil. Faecal samples were collected, and DNA was extracted using a silica-guanidine method. Five microsatellite loci were amplified using PCR with heterologous primers previously described for Lutra lutra (Linnaeus, 1758). Sixteen faecal samples out of 29 from Taim and 11 out of 14 from Vargas stream margins contained enough DNA for genetic analysis. A total of 49 different alleles were found at both localities, from which 18 were exclusively found in individuals from Taim and 17 were exclusives from Vargas individuals. The most common allele was the same at both locations for three loci (Lut715, Lut733, and Lut818). A high level of genetic diversity was found at both sites (NeTaim=4.1, HoTaim=0.299, HeTaim=0.681; NeVargas=4.9, HoVargas=0.355, HeVargas=0.724), being higher at the Vargas stream site. A high and significant level of heterozygote deficiency was observed at most loci according to the χ2 test. The homogeneity χ2 test (P<0.001) showed that there were significant differences in the allele frequencies between the two locations. Genotyping for more than one locus was possible in 81.5% of samples, from which only 37% were possible to genotype for more than three loci. A low degree of relatedness was found among individuals from Taim (R=0.055±0.310), but an even lower value of relatedness was found at the Vargas site (R= -0.285±0.440). The significant degree of differentiation (I=0.890; F ST=0.059) found between Taim and Vargas individuals suggests that there is more than one population of otters in the southern extreme of Brazil, which probably are associated with the water body systems found in this region, the Mirim and the Caiuvá/Flores/Mangueira Lagoons. The high genetic diversity and low relatedness found at the Vargas stream, lead us to believe that the Vargas stream may be acting as a corridor between these water bodies for otter dispersion.


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