Evaluating methods for purifying cyanobacterial cultures by qPCR and high-throughput Illumina sequencing

Silva, Karina Heck da; Machineski, Gabriela Silva; Alvarenga, Danillo Oliveira; Vaz, Marcelo Gomes Marçal Vieira; Varani, Alessandro de Mello; Fiore, Marli Fátima

doi:10.1016/j.mimet.2016.07.023

Cited by 23 publications

(16 citation statements)

References 37 publications

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“…Most research labs keep non-axenic, unicyanobacterial cultures for routine work, and only dedicate time for trying to achieve an axenic culture when a specific strain is scheduled to be studied in further detail, e.g., for genomic sequencing. Furthermore, traditional methods for evaluating axenity in cyanobacterial cultures may be misleading, as they check for the persistence of cultured bacteria while ignoring uncultured symbionts, causing a mixed culture to be mistaken for an axenic one (Heck et al, 2016). …”

Section: Non-axenic Cultures In Cyanobacteriologymentioning

confidence: 99%

A Metagenomic Approach to Cyanobacterial Genomics

2017

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Cyanobacteria, or oxyphotobacteria, are primary producers that establish ecological interactions with a wide variety of organisms. Although their associations with eukaryotes have received most attention, interactions with bacterial and archaeal symbionts have also been occurring for billions of years. Due to these associations, obtaining axenic cultures of cyanobacteria is usually difficult, and most isolation efforts result in unicyanobacterial cultures containing a number of associated microbes, hence composing a microbial consortium. With rising numbers of cyanobacterial blooms due to climate change, demand for genomic evaluations of these microorganisms is increasing. However, standard genomic techniques call for the sequencing of axenic cultures, an approach that not only adds months or even years for culture purification, but also appears to be impossible for some cyanobacteria, which is reflected in the relatively low number of publicly available genomic sequences of this phylum. Under the framework of metagenomics, on the other hand, cumbersome techniques for achieving axenic growth can be circumvented and individual genomes can be successfully obtained from microbial consortia. This review focuses on approaches for the genomic and metagenomic assessment of non-axenic cyanobacterial cultures that bypass requirements for axenity. These methods enable researchers to achieve faster and less costly genomic characterizations of cyanobacterial strains and raise additional information about their associated microorganisms. While non-axenic cultures may have been previously frowned upon in cyanobacteriology, latest advancements in metagenomics have provided new possibilities for in vitro studies of oxyphotobacteria, renewing the value of microbial consortia as a reliable and functional resource for the rapid assessment of bloom-forming cyanobacteria.

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Section: Non-axenic Cultures In Cyanobacteriologymentioning

confidence: 99%

A Metagenomic Approach to Cyanobacterial Genomics

2017

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“…Cells from the cyanobacterial culture were collected and processed as previously described (Heck et al, 2016). Total genomic DNA was extracted using the AxyPrep™ Bacterial Genomic DNA Miniprep Kit (Axygen Biosciences) according to manufacturer instructions.…”

Section: Dna Extraction Pcr Amplification and Sanger Sequencingmentioning

confidence: 99%

Biosynthesis of microcystin hepatotoxins in the cyanobacterial genus Fischerella

Silva

Alvarenga

Shishido

et al. 2018

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Microcystins (MCs) are serine/threonine phosphatase inhibitors synthesized by several members of the phylum Cyanobacteria. Mining the draft genome sequence of the nostocalean MC-producing Fischerella sp. strain CENA161 led to the identification of three contigs containing mcy genes. Subsequent PCR and Sanger sequencing allowed the assembling of its complete biosynthetic mcy gene cluster with 55,016 bases in length. The cluster encoding ten genes (mcyA-J) with a central bidirectional promoter was organized in a similar manner as found in other genera of nostocalean cyanobacteria. However, the nucleotide sequence of the mcy gene cluster of Fischerella sp. CENA161 showed significant differences from all the other MC-producing cyanobacterial genera, sharing only 85.2 to 74.1% identities. Potential MC variants produced by Fischerella sp. CENA161 were predicted by the analysis of the adenylation domain binding pockets and further investigated by LC-MS/MS analysis. To our knowledge, this study presents the first complete mcy cluster characterization from a strain of the genus Fischerella, providing new insight into the distribution and evolution of MCs in the phylum Cyanobacteria.

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“…While PCR-based methods can detect contaminants in genomic DNA, the methods are limited to specifically targeted contaminants and are not amenable to highly multiplexed applications (Heck et al, 2016;Marron et al, 2013). In contrast to these methods, shotgun metagenomic methods, though unable to assess contaminant viability, can be used to detect contaminants in both cell cultures and genomic DNA materials while only requiring the contaminant has sequencing reads differentiating it from the material strain.…”

Section: Introductionmentioning

confidence: 99%

Peer Review #1 of "Challenging a bioinformatic tool’s ability to detect microbial contaminants using in silico whole genome sequencing data (v0.1)"

Kumar¹

2017

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High sensitivity methods such as next generation sequencing and polymerase chain reaction (PCR) are adversely impacted by organismal and DNA contaminants. Current methods for detecting contaminants in microbial materials (genomic DNA and cultures) are not sensitive enough and require either a known or culturable contaminant. Whole genome sequencing (WGS) is a promising approach for detecting contaminants due to its sensitivity and lack of need for a priori assumptions about the contaminant. Prior to applying WGS, we must first understand its limitations for detecting contaminants and potential for false positives. Herein we demonstrate and characterize a WGS-based approach to detect organismal contaminants using an existing metagenomic taxonomic classification algorithm. Simulated WGS datasets from ten genera as individuals and binary mixtures of eight organisms at varying ratios were analyzed to evaluate the role of contaminant concentration and taxonomy on detection. For the individual genomes the false positive contaminants reported depended on the genus with Staphylococcus, Escherichia, and Shigella having the highest proportion of false positives. For nearly all binary mixtures the contaminant was detected in the in-silico datasets at the equivalent of 1 in 1,000 cells. Though F. tularensis was not detected in any of the simulated contaminant mixtures and Y. pestis was only detected at the equivalent of 1 in 10 cells. Once a WGS method for detecting contaminants is characterized, it can be applied to evaluate microbial material purity, in effort to ensure that contaminants in microbial materials used to validate pathogen detection assays, generate genome assemblies for database submission, and benchmark sequencing methods.

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Evaluating methods for purifying cyanobacterial cultures by qPCR and high-throughput Illumina sequencing

Cited by 23 publications

References 37 publications

A Metagenomic Approach to Cyanobacterial Genomics

A Metagenomic Approach to Cyanobacterial Genomics

Biosynthesis of microcystin hepatotoxins in the cyanobacterial genus Fischerella

Peer Review #1 of "Challenging a bioinformatic tool’s ability to detect microbial contaminants using in silico whole genome sequencing data (v0.1)"

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