scholarly journals Evaluation of methods for the reduction of contaminating host reads when performing shotgun metagenomic sequencing of the milk microbiome

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Min Yap ◽  
Conor Feehily ◽  
Calum J. Walsh ◽  
Mark Fenelon ◽  
Eileen F. Murphy ◽  
...  
Keyword(s):  
Human Milk ◽  
Sequencing Depth ◽  
Metagenomic Sequencing ◽  
Mock Community ◽  
Milk Samples ◽  
Optimal Classifier ◽  
Shotgun Metagenomic Sequencing ◽  
Functional Profiling ◽  
Microbial Dna

AbstractShotgun metagenomic sequencing is a valuable tool for the taxonomic and functional profiling of microbial communities. However, this approach is challenging in samples, such as milk, where a low microbial abundance, combined with high levels of host DNA, result in inefficient and uneconomical sequencing. Here we evaluate approaches to deplete host DNA or enrich microbial DNA prior to sequencing using three commercially available kits. We compared the percentage of microbial reads obtained from each kit after shotgun metagenomic sequencing. Using bovine and human milk samples, we determined that host depletion with the MolYsis complete5 kit significantly improved microbial sequencing depth compared to other approaches tested. Importantly, no biases were introduced. Additionally, the increased microbial sequencing depth allowed for further characterization of the microbiome through the generation of metagenome-assembled genomes (MAGs). Furthermore, with the use of a mock community, we compared three common classifiers and determined that Kraken2 was the optimal classifier for these samples. This evaluation shows that microbiome analysis can be performed on both bovine and human milk samples at a much greater resolution without the need for more expensive deep-sequencing approaches.

scholarly journals Evaluation of Methods to Optimise Shotgun Metagenomic Sequencing-Based Analysis of the Microbiome of Milk

2020 ◽  
Author(s):  
Min Yap ◽  
Conor Feehily ◽  
Calum J. Walsh ◽  
Mark Fenelon ◽  
Eileen F. Murphy ◽  
...  
Keyword(s):  
Human Milk ◽  
Sequencing Depth ◽  
Metagenomic Sequencing ◽  
Microbiome Analysis ◽  
Milk Samples ◽  
Shotgun Metagenomic Sequencing ◽  
Functional Profiling ◽  
Microbial Dna ◽  
Evaluation Of Methods

Abstract Background: Shotgun metagenomic sequencing is a valuable tool for the taxonomic and functional profiling of microbial communities. However, this approach is challenging in samples, such as milk, where a low microbial abundance, combined with high levels of host DNA, result in inefficient and uneconomical sequencing.Results: Here we evaluate approaches to deplete host DNA or enrich microbial DNA prior to sequencing using three commercially available kits. We compared the percentage of microbial reads obtained from each kit after shotgun metagenomic sequencing. Using bovine and human milk samples, we determined that host depletion with the MolYsis complete5 kit significantly improved microbial sequencing depth compared to other approaches. Importantly, no biases were introduced. Additionally, the increased microbial sequencing depth allowed for further characterization of the microbiome through the generation of metagenome-assembled genomes (MAGs). Conclusions: This evaluation shows that microbiome analysis can be performed on both bovine and human milk samples at a much greater resolution without the need for more expensive deep-sequencing approaches.

scholarly journals Targeted high-resolution taxonomic identification of Bifidobacterium longum subsp. infantis using human milk oligosaccharide metabolizing genes

2021 ◽  
Author(s):  
Lauren Tso ◽  
Kevin S Bonham ◽  
Alyssa Fishbein ◽  
Sophie Rowland ◽  
Vanja Klepac-Ceraj ◽  
...  
Keyword(s):  
Human Milk ◽  
Bifidobacterium Longum ◽  
Gene Clusters ◽  
Species Group ◽  
Human Breast Milk ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Specific Primers ◽  
Breastfed Infants ◽  
Shotgun Metagenomic Sequencing

Bifidobacterium longum subsp. infantis (B. infantis) is one of few microorganisms capable of metabolizing human breast milk and is a pioneer colonizer in the guts of breastfed infants. One current challenge is differentiating B. infantis from its close relatives, B. longum and B. suis, by molecular methods. These two organisms are classified in the same species group as B. infantis but do not share the ability to metabolize human milk oligosaccharides (HMOs). Here, we compared HMO-metabolizing genes across different Bifidobacterium genomes to develop B. infantis specific primers and determine if they alone can be used to quickly characterize B. infantis with shotgun metagenomic sequencing data. We showed that B. infantis is uniquely identified by the presence of five HMO-metabolizing gene clusters, used this characterization to test for its prevalence in infants, and validated the results using the B. infantis-specific primers. By examining stool samples from a cohort of US children and pregnant women using shotgun metagenomic sequencing, we observed that only 18 of 204 (8.8%) of children under 2 years old harbored B. infantis. These results highlight the importance of developing and improving approaches to identify B. infantis. A more accurate characterization may provide insights into regional differences of B. infantis prevalence in infant gut microbiota.

scholarly journals An Alignment-Free “Metapeptide” Strategy for Metaproteomic Characterization of Microbiome Samples Using Shotgun Metagenomic Sequencing

2016 ◽  
Vol 15 (8) ◽  
pp. 2697-2705 ◽  
Author(s):  
Damon H. May ◽  
Emma Timmins-Schiffman ◽  
Molly P. Mikan ◽  
H. Rodger Harvey ◽  
Elhanan Borenstein ◽  
...  
Keyword(s):  
Metagenomic Sequencing ◽  
Alignment Free ◽  
Shotgun Metagenomic Sequencing

scholarly journals Comparative analysis of 16S rRNA gene and metagenome sequencing in pediatric gut microbiomes

2021 ◽  
Author(s):  
Danielle Peterson ◽  
Kevin S. Bonham ◽  
Sophie Rowland ◽  
Cassandra W. Pattanayak ◽  
Vanja Klepac-Ceraj ◽  
...  
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
Gut Microbiome ◽  
Age Groups ◽  
Alpha Diversity ◽  
Sequencing Depth ◽  
Human Infant ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Shotgun Metagenomic Sequencing

AbstractThe colonization of the human gut microbiome begins at birth, and, over time, these microbial communities become increasingly complex. Most of what we currently know about the human microbiome, especially in early stages of development, was described using culture-independent sequencing methods that allow us to identify the taxonomic composition of microbial communities using genomic techniques, such as amplicon or shotgun metagenomic sequencing. Each method has distinct tradeoffs, but there has not been a direct comparison of the utility of these methods in stool samples from very young children, which have different features than those of adults. We compared the effects of profiling the human infant gut microbiome with 16S rRNA amplicon versus shotgun metagenomic sequencing techniques in 130 fecal samples; younger than 15, 15-30, and older than 30 months of age. We demonstrate that observed changes in alpha-diversity and beta-diversity with age occur to similar extents using both profiling methods. We also show that 16S rRNA profiling identified a larger number of genera and we find several genera that are missed or underrepresented by each profiling method. We present the link between alpha diversity and shotgun metagenomic sequencing depth for children of different ages. These findings provide a guide for selecting an appropriate method and sequencing depth for the three studied age groups.

scholarly journals Molecular characterization of methicillin-resistant and methicillin-sensitive Staphylococcus aureus isolates from human milk samples in Brazil

2020 ◽  
Vol 51 (4) ◽  
pp. 1813-1817
Author(s):  
Jéssica Bomfim de Almeida ◽  
Suzi Pacheco de Carvalho ◽  
Lucas S. C. da Silva ◽  
Yasmin M. F. S. Andrade ◽  
Raiane Cardoso Chamon ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Human Milk ◽  
Molecular Characterization ◽  
Methicillin Resistant ◽  
Milk Samples

scholarly journals Metagenomics-Based Proficiency Test of Smoked Salmon Spiked with a Mock Community

2020 ◽  
Vol 8 (12) ◽  
pp. 1861
Author(s):  
Claudia Sala ◽  
Hanne Mordhorst ◽  
Josephine Grützke ◽  
Annika Brinkmann ◽  
Thomas N. Petersen ◽  
...  
Keyword(s):  
Proficiency Test ◽  
Metagenomic Sequencing ◽  
Food Matrix ◽  
Community Members ◽  
Mock Community ◽  
Sequencing Platform ◽  
Shotgun Metagenomic Sequencing ◽  
Smoked Salmon ◽  
Genomic Tool ◽  
Wet Lab

An inter-laboratory proficiency test was organized to assess the ability of participants to perform shotgun metagenomic sequencing of cold smoked salmon, experimentally spiked with a mock community composed of six bacteria, one parasite, one yeast, one DNA, and two RNA viruses. Each participant applied its in-house wet-lab workflow(s) to obtain the metagenomic dataset(s), which were then collected and analyzed using MG-RAST. A total of 27 datasets were analyzed. Sample pre-processing, DNA extraction protocol, library preparation kit, and sequencing platform, influenced the abundance of specific microorganisms of the mock community. Our results highlight that despite differences in wet-lab protocols, the reads corresponding to the mock community members spiked in the cold smoked salmon, were both detected and quantified in terms of relative abundance, in the metagenomic datasets, proving the suitability of shotgun metagenomic sequencing as a genomic tool to detect microorganisms belonging to different domains in the same food matrix. The implementation of standardized wet-lab protocols would highly facilitate the comparability of shotgun metagenomic sequencing dataset across laboratories and sectors. Moreover, there is a need for clearly defining a sequencing reads threshold, to consider pathogens as detected or undetected in a food sample.

scholarly journals Metagenomic Information Recovery from Human Stool Samples Is Influenced by Sequencing Depth and Profiling Method

Genes ◽  
2020 ◽  
Vol 11 (11) ◽  
pp. 1380
Author(s):  
Tasha M. Santiago-Rodriguez ◽  
Aaron Garoutte ◽  
Emmase Adams ◽  
Waleed Nasser ◽  
Matthew C. Ross ◽  
...  
Keyword(s):  
Gene Mapping ◽  
Marker Gene ◽  
Species Level ◽  
Sequencing Depth ◽  
Gene Content ◽  
Metagenomic Sequencing ◽  
16S Sequencing ◽  
Shotgun Metagenomic Sequencing ◽  
Stool Samples ◽  
Human Stool

Sequencing of the 16S rRNA gene (16S) has long been a go-to method for microbiome characterization due to its accessibility and lower cost compared to shotgun metagenomic sequencing (SMS). However, 16S sequencing rarely provides species-level resolution and cannot provide direct assessment of other taxa (e.g., viruses and fungi) or functional gene content. Shallow shotgun metagenomic sequencing (SSMS) has emerged as an approach to bridge the gap between 16S sequencing and deep metagenomic sequencing. SSMS is cost-competitive with 16S sequencing, while also providing species-level resolution and functional gene content insights. In the present study, we evaluated the effects of sequencing depth on marker gene-mapping- and alignment-based annotation of bacteria in healthy human stool samples. The number of identified taxa decreased with lower sequencing depths, particularly with the marker gene-mapping-based approach. Other annotations, including viruses and pathways, also showed a depth-dependent effect on feature recovery. These results refine the understanding of the suitability and shortcomings of SSMS, as well as annotation tools for metagenomic analyses in human stool samples. Results may also translate to other sample types and may open the opportunity to explore the effect of sequencing depth and annotation method.

scholarly journals Characterization of SARS-CoV-2 RNA, Antibodies, and Neutralizing Capacity in Milk Produced by Women with COVID-19

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ryan M. Pace ◽  
Janet E. Williams ◽  
Kirsi M. Järvinen ◽  
Mandy B. Belfort ◽  
Christina D. W. Pace ◽  
...  
Keyword(s):  
Human Milk ◽  
Cross Sectional ◽  
Considerable Uncertainty ◽  
Milk Samples ◽  
Neutralizing Capacity ◽  
Specific Igg ◽  
Mother To Child ◽  
Do So

ABSTRACT Whether mother-to-infant SARS-CoV-2 transmission can occur during breastfeeding and, if so, whether the benefits of breastfeeding outweigh this risk during maternal COVID-19 illness remain important questions. Using RT-qPCR, we did not detect SARS-CoV-2 RNA in any milk sample (n = 37) collected from 18 women following COVID-19 diagnosis. Although we detected evidence of viral RNA on 8 out of 70 breast skin swabs, only one was considered a conclusive positive result. In contrast, 76% of the milk samples collected from women with COVID-19 contained SARS-CoV-2-specific IgA, and 80% had SARS-CoV-2-specific IgG. In addition, 62% of the milk samples were able to neutralize SARS-CoV-2 infectivity in vitro, whereas milk samples collected prior to the COVID-19 pandemic were unable to do so. Taken together, our data do not support mother-to-infant transmission of SARS-CoV-2 via milk. Importantly, milk produced by infected mothers is a beneficial source of anti-SARS-CoV-2 IgA and IgG and neutralizes SARS-CoV-2 activity. These results support recommendations to continue breastfeeding during mild-to-moderate maternal COVID-19 illness. IMPORTANCE Results from prior studies assaying human milk for the presence of SARS-CoV-2, the causative virus of COVID-19, have suggested milk may act as a potential vehicle for mother-to-child transmission. Most previous studies are limited because they followed only a few participants, were cross-sectional, and/or failed to report how milk was collected and/or analyzed. As such, considerable uncertainty remains regarding whether human milk is capable of transmitting SARS-CoV-2 from mother to child. Here, we report that repeated milk samples collected from 18 women following COVID-19 diagnosis did not contain SARS-CoV-2 RNA; however, risk of transmission via breast skin should be further evaluated. Importantly, we found that milk produced by infected mothers is a source of anti-SARS-CoV-2 IgA and IgG and neutralizes SARS-CoV-2 activity. These results support recommendations to continue breastfeeding during mild-to-moderate maternal COVID-19 illness as milk likely provides specific immunologic benefits to infants.

scholarly journals Bark from avocado trees of different geographic locations have consistent microbial communities

2020 ◽  
Author(s):  
Eneas Aguirre-von-Wobeser ◽  
Alexandro Alonso-Sánchez ◽  
Alfonso Méndez-Bravo ◽  
Luis Alberto Villanueva Espino ◽  
Frédérique Reverchon
Keyword(s):  
Microbial Communities ◽  
Persea Americana ◽  
Microbial Colonization ◽  
Taxonomic Structure ◽  
Metagenomic Sequencing ◽  
Important Species ◽  
Pests And Diseases ◽  
Shotgun Metagenomic Sequencing ◽  
Taxonomic Groups

AbstractBark is a permanent surface for microbial colonization at the interface of trees and the surrounding air. However, little is known about the microbial communities harbored on these tissues. Studies on bark microbial ecology show a dominance of bacteria from a few phyla. Bark microbial communities of avocado (Persea americana) could have implications for tree health, as a first barrier for defense against certain pests and diseases in this economically important species. We used shotgun metagenomic sequencing to analyze the bark microbial communities of avocado trees from two orchards, and compared one of them to rhizospheric soil. Our results show that the microbial communities of avocado bark have a well-defined taxonomic structure, with consistent patterns of abundance of bacteria, fungi and archaea, even in trees from two different locations. Bacteria in avocado bark were dominated by Proteobacteria (particularly Alphaproteobacteria), Actinobacteria and Bacteroidetes, consistently with bark communities in other trees. Fungal members were dominated by Ascomycota and Basidiomycota, while most Archaea in bark were Euryarchaeota. We can conclude that avocado bark is a well-defined environment, providing niches for specific taxonomic groups. The present in-depth characterization of bark microbial communities can form a basis for their future manipulation for agronomical purposes.

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