scholarly journals A Two-Step PCR Protocol Enabling Flexible Primer Choice and High Sequencing Yield for Illumina MiSeq Meta-Barcoding

Agronomy ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1274
Author(s):  
Ko-Hsuan Chen ◽  
Reid Longley ◽  
Gregory Bonito ◽  
Hui-Ling Liao
Keyword(s):  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Agricultural Systems ◽  
Specific Primers ◽  
Community Discovery ◽  
16S Ribosomal Dna ◽  
Genomic Regions ◽  
Internal Transcribed Spacer Rdna ◽  
Fungal Its ◽  
Illumina Amplicon Sequencing

High-throughput amplicon sequencing that primarily targets the 16S ribosomal DNA (rDNA) (for bacteria and archaea) and the Internal Transcribed Spacer rDNA (for fungi) have facilitated microbial community discovery across diverse environments. A three-step PCR that utilizes flexible primer choices to construct the library for Illumina amplicon sequencing has been applied to several studies in forest and agricultural systems. The three-step PCR protocol, while producing high-quality reads, often yields a large number (up to 46%) of reads that are unable to be assigned to a specific sample according to its barcode. Here, we improve this technique through an optimized two-step PCR protocol. We tested and compared the improved two-step PCR meta-barcoding protocol against the three-step PCR protocol using four different primer pairs (fungal ITS: ITS1F-ITS2 and ITS1F-ITS4, and bacterial 16S: 515F-806R and 341F-806R). We demonstrate that the sequence quantity and recovery rate were significantly improved with the two-step PCR approach (fourfold more read counts per sample; determined reads ≈90% per run) while retaining high read quality (Q30 > 80%). Given that synthetic barcodes are incorporated independently from any specific primers, this two-step PCR protocol can be broadly adapted to different genomic regions and organisms of scientific interest.

scholarly journals Identifying Hidden Viable Bacterial Taxa in Tropical Forest Soils Using Amplicon Sequencing of Enrichment Cultures

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 569
Author(s):  
Chakriya Sansupa ◽  
Sara Fareed Mohamed Wahdan ◽  
Terd Disayathanoowat ◽  
Witoon Purahong
Keyword(s):  
Bacterial Community ◽  
Forest Soil ◽  
Culture Media ◽  
Enrichment Culture ◽  
Sequence Similarity ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Soil Samples ◽  
Enrichment Cultures ◽  
Total Community

This study aims to estimate the proportion and diversity of soil bacteria derived from eDNA-based and culture-based methods. Specifically, we used Illumina Miseq to sequence and characterize the bacterial communities from (i) DNA extracted directly from forest soil and (ii) DNA extracted from a mixture of bacterial colonies obtained by enrichment cultures on agar plates of the same forest soil samples. The amplicon sequencing of enrichment cultures allowed us to rapidly screen a culturable community in an environmental sample. In comparison with an eDNA community (based on a 97% sequence similarity threshold), the fact that enrichment cultures could capture both rare and abundant bacterial taxa in forest soil samples was demonstrated. Enrichment culture and eDNA communities shared 2% of OTUs detected in total community, whereas 88% of enrichment cultures community (15% of total community) could not be detected by eDNA. The enrichment culture-based methods observed 17% of the bacteria in total community. FAPROTAX functional prediction showed that the rare and unique taxa, which were detected with the enrichment cultures, have potential to perform important functions in soil systems. We suggest that enrichment culture-based amplicon sequencing could be a beneficial approach to evaluate a cultured bacterial community. Combining this approach together with the eDNA method could provide more comprehensive information of a bacterial community. We expected that more unique cultured taxa could be detected if further studies used both selective and non-selective culture media to enrich bacteria at the first step.

DNA extraction from concrete v2

2021 ◽  
Author(s):  
Anders Kiledal ◽  
Julia A Maresca
Keyword(s):  
Dna Extraction ◽  
Dental Plaque ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Metagenomic Sequencing ◽  
Sample Analysis ◽  
Larger Sample ◽  
Laboratory Contamination ◽  
Biomass Sample ◽  
Over Time

This is a protocol for extracting DNA from concrete, based on the protocol developed by L. S. Weyrich, et al. for extraction of DNA from ancient calcified dental plaque. We have scaled it up for larger sample sizes and made some additional modifications for the chemistry of concrete. DNA extracted using this method is suitable for metagenomic sequencing by Illumina MiSeq and NextSeq, as well as amplicon sequencing. This protocol should yield 10 ng to 5 μg DNA per 10 g of concrete, depending on the age and integrity of the sample. Reference: L. S. Weyrich et al., Laboratory contamination over time during low-biomass sample analysis. Mol. Ecol. Resour. 19, 982–996 (2019).

scholarly journals A novel proof of concept for capturing the diversity of endophytic fungi preserved in herbarium specimens

2018 ◽  
Vol 374 (1763) ◽  
pp. 20170395 ◽  
Author(s):  
Barnabas H. Daru ◽  
Elizabeth A. Bowman ◽  
Donald H. Pfister ◽  
A. Elizabeth Arnold
Keyword(s):  
Species Interactions ◽  
Fungal Endophytes ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Herbarium Specimens ◽  
Proof Of Concept ◽  
Ledum Palustre ◽  
Surface Contaminants ◽  
Symptomless Plant ◽  
Miseq Platform

Herbarium specimens represent important records of morphological and genetic diversity of plants that inform questions relevant to global change, including species distributions, phenology and functional traits. It is increasingly appreciated that plant microbiomes can influence these aspects of plant biology, but little is known regarding the historic distribution of microbes associated with plants collected in the pre-molecular age. If microbiomes can be observed reliably in herbarium specimens, researchers will gain a new lens with which to examine microbial ecology, evolution, species interactions. Here, we describe a method for accessing historical plant microbiomes from preserved herbarium specimens, providing a proof of concept using two plant taxa from the imperiled boreal biome ( Andromeda polifolia and Ledum palustre subsp . groenlandicum, Ericaceae). We focus on fungal endophytes, which occur within symptomless plant tissues such as leaves. Through a three-part approach (i.e. culturing, cloning and next-generation amplicon sequencing via the Illumina MiSeq platform, with extensive controls), we examined endophyte communities in dried, pressed leaves that had been processed as regular herbarium specimens and stored at room temperature in a herbarium for four years . We retrieved only one endophyte in culture, but cloning and especially the MiSeq analysis revealed a rich community of foliar endophytes. The phylogenetic distribution and diversity of endophyte assemblages, especially among the Ascomycota, resemble endophyte communities from fresh plants collected in the boreal biome. We could distinguish communities of endophytes in each plant species and differentiate likely endophytes from fungi that could be surface contaminants. Taxa found by cloning were observed in the larger MiSeq dataset, but species richness was greater when subsets of the same tissues were evaluated with the MiSeq approach. Our findings provide a proof of concept for capturing endophyte DNA from herbarium specimens, supporting the importance of herbarium records as roadmaps for understanding the dynamics of plant-associated microbial biodiversity in the Anthropocene. This article is part of the theme issue ‘Biological collections for understanding biodiversity in the Anthropocene’.

scholarly journals Combination of Multiple Ligation-Dependent Probe Amplification and Illumina MiSeq Amplicon Sequencing for TSC1/TSC2 Gene Analyses in Patients with Tuberous Sclerosis Complex

2017 ◽  
Vol 19 (2) ◽  
pp. 265-276 ◽  
Author(s):  
Nur Farrah Dila Ismail ◽  
Abdul Qawee Rani ◽  
Nik Mohd Ariff Nik Abdul Malik ◽  
Chia Boon Hock ◽  
Siti Nabilahuda Mohd Azlan ◽  
...  
Keyword(s):  
Tuberous Sclerosis ◽  
Tuberous Sclerosis Complex ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Tsc2 Gene ◽  
Dependent Probe

scholarly journals UNCROSS: Filtering of high-frequency cross-talk in 16S amplicon reads

2016 ◽  
Author(s):  
Robert C. Edgar
Keyword(s):  
Ribosomal Rna ◽  
Cross Talk ◽  
High Frequency ◽  
Biological Diversity ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Illumina Gaiix ◽  
Microbial Metagenomics ◽  
Tumor Sequencing ◽  
Rna I

AbstractNext-generation amplicon sequencing is widely used for surveying biological diversity in applications such as microbial metagenomics, immune system repertoire analysis and targeted tumor sequencing of cancer-associated genes. In such studies, assignment of reads to incorrect samples (cross-talk) is a well-documented problem that is rarely considered in practice. By considering unexpected OTUs in artificial (mock) samples, I estimate that cross-talk occurred for ~2% of the reads in one Illumina GAIIx run and eleven Illumina MiSeq runs targeting 16S ribosomal RNA. I also describe UNCROSS, an algorithm for detecting and filtering cross-talk in OTU tables.

scholarly journals Bacterial community associated with worker honeybees (Apis mellifera) affected by European foulbrood

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3816 ◽  
Author(s):  
Tomas Erban ◽  
Ondrej Ledvinka ◽  
Martin Kamler ◽  
Bronislava Hortova ◽  
Marta Nesvorna ◽  
...  
Keyword(s):  
Apis Mellifera ◽  
Clinical Symptoms ◽  
Clinical Signs ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Melissococcus Plutonius ◽  
European Foulbrood ◽  
Bacterial Groups ◽  
V3 Region

BackgroundMelissococcus plutoniusis an entomopathogenic bacterium that causes European foulbrood (EFB), a honeybee (Apis melliferaL.) disease that necessitates quarantine in some countries. In Czechia, positive evidence of EFB was absent for almost 40 years, until an outbreak in the Krkonose Mountains National Park in 2015. This occurrence of EFB gave us the opportunity to study the epizootiology of EFB by focusing on the microbiome of honeybee workers, which act as vectors of honeybee diseases within and between colonies.MethodsThe study included worker bees collected from brood combs of colonies (i) with no signs of EFB (EFB0), (ii) without clinical symptoms but located at an apiary showing clinical signs of EFB (EFB1), and (iii) with clinical symptoms of EFB (EFB2). In total, 49 samples from 27 honeybee colonies were included in the dataset evaluated in this study. Each biological sample consisted of 10 surface-sterilized worker bees processed for DNA extraction. All subjects were analyzed using conventional PCR and by metabarcoding analysis based on the 16S rRNA gene V1–V3 region, as performed through Illumina MiSeq amplicon sequencing.ResultsThe bees from EFB2 colonies with clinical symptoms exhibited a 75-fold-higher incidence ofM. plutoniusthan those from EFB1 asymptomatic colonies.Melissococcus plutoniuswas identified in all EFB1 colonies as well as in some of the control colonies. The proportions ofFructobacillus fructosus,Lactobacillus kunkeei,Gilliamella apicola,Frischella perrara, andBifidobacterium coryneformewere higher in EFB2 than in EFB1, whereasLactobacillus melliswas significantly higher in EFB2 than in EFB0.Snodgrassella alviandL. melliventris,L. helsingborgensisand,L. kullabergensisexhibited higher proportion in EFB1 than in EFB2 and EFB0. The occurrence ofBartonella apisandCommensalibacter intestiniwere higher in EFB0 than in EFB2 and EFB1.Enterococcus faecalisincidence was highest in EFB2.ConclusionsHigh-throughput Illumina sequencing permitted a semi-quantitative analysis of the presence ofM. plutoniuswithin the honeybee worker microbiome. The results of this study indicate that worker bees from EFB-diseased colonies are capable of transmittingM. plutoniusdue to the greatly increased incidence of the pathogen. The presence ofM. plutoniussequences in control colonies supports the hypothesis that this pathogen exists in an enzootic state. The bacterial groups synergic to both the colonies with clinical signs of EFB and the EFB-asymptomatic colonies could be candidates for probiotics. This study confirms thatE. faecalisis a secondary invader toM. plutonius; however, other putative secondary invaders were not identified in this study.

scholarly journals Genomic characterization of Vibrio parahaemolyticus from Pacific white shrimp and rearing water in Malaysia reveals novel sequence types and structural variation in genomic regions containing the Photorhabdus insect-related (Pir) toxin-like genes

2019 ◽  
Vol 366 (17) ◽  
Author(s):  
Chrystine Zou Yi Yan ◽  
Christopher M Austin ◽  
Qasim Ayub ◽  
Sadequr Rahman ◽  
Han Ming Gan
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Illumina Miseq ◽  
Pacific White Shrimp ◽  
White Shrimp ◽  
Phylogenomic Analysis ◽  
Genomic Characterization ◽  
Genomic Resource ◽  
Acute Hepatopancreatic Necrosis Disease ◽  
Genomic Regions

ABSTRACT The Malaysian and global shrimp aquaculture production has been significantly impacted by acute hepatopancreatic necrosis disease (AHPND) typically caused by Vibrio parahaemolyticus harboring the pVA plasmid containing the pirAVp and pirBVp genes, which code for Photorhabdus insect-related (Pir) toxin. The limited genomic resource for V. parahaemolyticus strains from Malaysian aquaculture farms precludes an in-depth understanding of their diversity and evolutionary relationships. In this study, we isolated shrimp-associated and environmental (rearing water) V. parahaemolyticus from three aquaculture farms located in Northern and Central Malaysia followed by whole-genome sequencing of 40 randomly selected isolates on the Illumina MiSeq. Phylogenomic analysis and multilocus sequence typing (MLST) reveal distinct lineages of V. parahaemolyticus that harbor the pirABVp genes. The recovery of pVA plasmid backbone devoid of pirAVp or pirABVp in some V. parahaemolyticus isolates suggests that the toxin genes are prone to deletion. The new insight gained from phylogenomic analysis of Asian V. parahaemolyticus, in addition to the observed genomic instability of pVa plasmid, will have implications for improvements in aquaculture practices to diagnose, treat or limit the impacts of this disease.

scholarly journals PSVI-16 Fecal microbiome of nursery pigs fed phytogenics and antibiotics

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 203-203
Author(s):  
Huyen Tran ◽  
Timothy J Johnson
Keyword(s):  
Beta Diversity ◽  
Sequence Data ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Fecal Microbiome ◽  
Nursery Pigs ◽  
Alpha And Beta Diversity ◽  
Family Level ◽  
Dietary Treatments

Abstract The objective of this study was to evaluate effects of feeding two phytogenic products (PHY1 and PHY2; blends of essential oils and plant extracts) in diets with or without antibiotics (AureoMix S 10-10; AB) on fecal microbiome of nursery pigs. A total of 400 nursery pigs (6.8 kg BW; 20 d of age) were fed one of the six dietary treatments (9 pens/treatment), including: control (0% AB; 0% phytogenics), 0.5% AB, phytogenics (0.02% PHY1 or 0.03% PHY2) or the combination of phytogenic and AB (PHY1 x AB or PHY2 x AB). On d 46 postweaning, 48 fecal samples were collected (1 pig/pen; 7–9 pigs/treatment) and were subjected to the analyses of microbial communities by using 16S rRNA V4 amplicon sequencing with Illumina MiSeq. The sequence data were analyzed by using Qiime and the rarefied OTU table was submitted to Calypso to evaluate the alpha and beta diversity, taxonomic classification, and the differential taxa associated to the dietary treatments. There were differences among treatments on alpha diversity, where the control and PHY2 pigs had lower OTU richness (P = 0.05) and chao1 index (P < 0.10) compared to pigs fed AB alone or AB with phytogenics. There were also differences among treatments on microbial beta diversity of pigs (P < 0.01). The most abundant phyla included Firmicute, Bacteroidetes, Actinobacteria, Tenericutes, Proteobacteria, Spirochaetes, and TM7. At family level, pigs fed AB had greater Ruminococcaceae compared to the control, but lower Coriobacteriaceae and Erysipelotrichaceae compared to PHY1 or PHY2 group (P < 0.05). Feature selection by LEfSe indicated that dominant genus associated to AB treatment was Unclassified RF39, while dominant genera associated to PHY2 treatment were Cantenibacterium, unclassified Coriobacteriaceae, Blautia, Eubacterium, and Collinsella. In conclusion, feeding AB and phytogenic products had different impacts on the fecal bacteria of nursery pigs.

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