More Like this

1. Using the Microbiome Amplification Preference Tool (MAPT) to Reveal Medicago sativa -Associated Eukaryotic Microbes

   https://doi.org/10.1094/PBIOMES-02-20-0022-R  

   Moccia, Katherine; Papoulis, Spiridon; Willems, Andrew; Marion, Zachary; Fordyce, James A.; Lebeis, Sarah L. (January 2020, Phytobiomes Journal)

   null (Ed.)

   Although our understanding of the microbial diversity found within a given system expands as amplicon sequencing improves, technical aspects still drastically affect which members can be detected. Compared with prokaryotic members, the eukaryotic microorganisms associated with a host are understudied due to their underrepresentation in ribosomal databases, lower abundance compared with bacterial sequences, and higher ribosomal gene identity to their eukaryotic host. Peptide nucleic acid (PNA) blockers are often designed to reduce amplification of host DNA. Here we present a tool for PNA design called the Microbiome Amplification Preference Tool (MAPT). We examine the effectiveness of a PNA designed to block genomic Medicago sativa DNA (gPNA) compared with unrelated surrounding plants from the same location. We applied mitochondrial PNA and plastid PNA to block the majority of DNA from plant mitochondria and plastid 16S ribosomal RNA genes, as well as the novel gPNA. Until now, amplifying both eukaryotic and prokaryotic reads using 515F-Y and 926R has not been applied to a host. We investigate the efficacy of this gPNA using three approaches: (i) in silico prediction of blocking potential in MAPT, (ii) amplicon sequencing with and without the addition of PNAs, and (iii) comparison with cultured fungal representatives. When gPNA is added during amplicon library preparation, the diversity of unique eukaryotic amplicon sequence variants present in M. sativa increases. We provide a layered examination of the costs and benefits of using PNAs during sequencing. The application of MAPT enables scientists to design PNAs specifically to enable capturing greater diversity in their system. 

   more » « less
2. NifH gene amplicon sequencing and metagenomic approaches are complementary in assessing diazotroph diversity

   https://doi.org/10.1093/ismeco/ycaf038  

   Cheung, Shunyan; Morando, Michael; Magasin, Jonathan; Cornejo-Castillo, Francisco_M; Zehr, Jonathan_P; Turk-Kubo, Kendra_A (February 2025, ISME Communications)

   Abstract Exploring the diversity of diazotrophs is key to understanding their role in supplying fixed nitrogen that supports marine productivity. A nested PCR assay using the universal primer set nifH1-nifH4, which targets the nitrogenase (nifH) gene, is a widely used approach for studying marine diazotrophs by amplicon sequencing. Metagenomics, direct sequencing of DNA without PCR, has provided complementary views of the diversity of marine diazotrophs. A significant fraction of the metagenome-derived nifH sequences (e.g. Planctomycete- and Proteobacteria-affiliated) were reported to have nucleotide mismatches with the nifH1-nifH4 primers, leading to the suggestion that nifH amplicon sequencing does not detect specific diazotrophic taxa and underrepresents diazotroph diversity. Here, we report that these mismatches are mostly located in a single-base at the 5′-end of the nifH4 primer, which does not impact detection of the nifH genes. This is demonstrated by the presence of nifH genes that contain the nucleotide mismatches in a recent compilation of global ocean nifH amplicon datasets, with high relative abundances detected in a variety of samples. While the metagenome- and metatranscriptome-derived nifH genes accounted for 4.4% of the total amplicon sequence variants from the global ocean nifH amplicon database, the corresponding amplicon sequence variants can have high relative abundances (accounting for 47% of the reads in the database). These analyses underscore that nifH amplicon sequencing using the nifH1-nifH4 primers is an important tool for studying diversity of marine diazotrophs, particularly as a complement to metagenomics which can provide taxonomic and metabolic information for some dominant groups. 

   more » « less
3. OGUs enable effective, phylogeny-aware analysis of even shallow metagenome community structures

   https://doi.org/2021.04.04.438427  

   Qiyun Zhu; Shi Huang; Antonio Gonzalez; Imran McGrath; Daniel McDonald; Niina Haiminen; George Armstrong; Yoshiki Vázquez-Baeza; Julian Yu; Justin Kuczynski; et al (April 2021, bioRxiv)

   We introduce Operational Genomic Unit (OGU), a metagenome analysis strategy that directly exploits sequence alignment hits to individual reference genomes as the minimum unit for assessing the diversity of microbial communities and their relevance to environmental factors. This approach is independent from taxonomic classification, granting the possibility of maximal resolution of community composition, and organizes features into an accurate hierarchy using a phylogenomic tree. The outputs are suitable for contemporary analytical protocols for community ecology, differential abundance and supervised learning while supporting phylogenetic methods, such as UniFrac and phylofactorization, that are seldomly applied to shotgun metagenomics despite being prevalent in 16S rRNA gene amplicon studies. As demonstrated in one synthetic and two real-world case studies, the OGU method produces biologically meaningful patterns from microbiome datasets. Such patterns further remain detectable at very low metagenomic sequencing depths. Compared with taxonomic unit-based analyses implemented in currently adopted metagenomics tools, and the analysis of 16S rRNA gene amplicon sequence variants, this method shows superiority in informing biologically relevant insights, including stronger correlation with body environment and host sex on the Human Microbiome Project dataset, and more accurate prediction of human age by the gut microbiomes in the Finnish population. We provide Woltka, a bioinformatics tool to implement this method, with full integration with the QIIME 2 package and the Qiita web platform, to facilitate OGU adoption in future metagenomics studies. Importance Shotgun metagenomics is a powerful, yet computationally challenging, technique compared to 16S rRNA gene amplicon sequencing for decoding the composition and structure of microbial communities. However, current analyses of metagenomic data are primarily based on taxonomic classification, which is limited in feature resolution compared to 16S rRNA amplicon sequence variant analysis. To solve these challenges, we introduce Operational Genomic Units (OGUs), which are the individual reference genomes derived from sequence alignment results, without further assigning them taxonomy. The OGU method advances current read-based metagenomics in two dimensions: (i) providing maximal resolution of community composition while (ii) permitting use of phylogeny-aware tools. Our analysis of real-world datasets shows several advantages over currently adopted metagenomic analysis methods and the finest-grained 16S rRNA analysis methods in predicting biological traits. We thus propose the adoption of OGU as standard practice in metagenomic studies. 

   more » « less
4. Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case Study With Reef-Building Corals

   https://doi.org/10.3389/fmicb.2021.740932  

   Epstein, Hannah E.; Hernandez-Agreda, Alejandra; Starko, Samuel; Baum, Julia K.; Vega Thurber, Rebecca (November 2021, Frontiers in Microbiology)

   16S rRNA gene profiling (amplicon sequencing) is a popular technique for understanding host-associated and environmental microbial communities. Most protocols for sequencing amplicon libraries follow a standardized pipeline that can differ slightly depending on laboratory facility and user. Given that the same variable region of the 16S gene is targeted, it is generally accepted that sequencing output from differing protocols are comparable and this assumption underlies our ability to identify universal patterns in microbial dynamics through meta-analyses. However, discrepant results from a combined 16S rRNA gene dataset prepared by two labs whose protocols differed only in DNA polymerase and sequencing platform led us to scrutinize the outputs and challenge the idea of confidently combining them for standard microbiome analysis. Using technical replicates of reef-building coral samples from two species, Montipora aequituberculata and Porites lobata , we evaluated the consistency of alpha and beta diversity metrics between data resulting from these highly similar protocols. While we found minimal variation in alpha diversity between platform, significant differences were revealed with most beta diversity metrics, dependent on host species. These inconsistencies persisted following removal of low abundance taxa and when comparing across higher taxonomic levels, suggesting that bacterial community differences associated with sequencing protocol are likely to be context dependent and difficult to correct without extensive validation work. The results of this study encourage caution in the statistical comparison and interpretation of studies that combine rRNA gene sequence data from distinct protocols and point to a need for further work identifying mechanistic causes of these observed differences. 

   more » « less
5. A CRISPR ‐based strategy for targeted sequencing in biodiversity science

   https://doi.org/10.1111/1755-0998.13920  

   Littleford‐Colquhoun, Bethan; Kartzinel, Tyler R. (December 2023, Molecular Ecology Resources)

   Abstract Many applications in molecular ecology require the ability to match specific DNA sequences from single‐ or mixed‐species samples with a diagnostic reference library. Widely used methods for DNA barcoding and metabarcoding employ PCR and amplicon sequencing to identify taxa based on target sequences, but the target‐specific enrichment capabilities of CRISPR‐Cas systems may offer advantages in some applications. We identified 54,837 CRISPR‐Cas guide RNAs that may be useful for enriching chloroplast DNA across phylogenetically diverse plant species. We tested a subset of 17 guide RNAs in vitro to enrich plant DNA strands ranging in size from diagnostic DNA barcodes of 1,428 bp to entire chloroplast genomes of 121,284 bp. We used an Oxford Nanopore sequencer to evaluate sequencing success based on both single‐ and mixed‐species samples, which yielded mean chloroplast sequence lengths of 2,530–11,367 bp, depending on the experiment. In comparison to mixed‐species experiments, single‐species experiments yielded more on‐target sequence reads and greater mean pairwise identity between contigs and the plant species' reference genomes. But nevertheless, these mixed‐species experiments yielded sufficient data to provide ≥48‐fold increase in sequence length and better estimates of relative abundance for a commercially prepared mixture of plant species compared to DNA metabarcoding based on the chloroplasttrnL‐P6 marker. Prior work developed CRISPR‐based enrichment protocols for long‐read sequencing and our experiments pioneered its use for plant DNA barcoding and chloroplast assemblies that may have advantages over workflows that require PCR and short‐read sequencing. Future work would benefit from continuing to develop in vitro and in silico methods for CRISPR‐based analyses of mixed‐species samples, especially when the appropriate reference genomes for contig assembly cannot be known a priori. 

   more » « less