Development and Bias Assessment of a Method for Targeted Metagenomic Sequencing of Marine Cyanobacteria

Abstract

Prochlorococcus and Synechococcus are the most abundant photosynthetic organisms in oligotrophic waters and responsible for a significant percentage of the earth's primary production. Here we developed a method for metagenomic sequencing of sorted Prochlorococcus and Synechococcus populations using a transposon-based library preparation technique. First, we observed that the cell lysis technique and associated amount of input DNA had an important role in determining the DNA library quality. Second, we found that our transposon-based method provided a more even coverage distribution and matched more sequences of a reference genome than multiple displacement amplification, a commonly used method for metagenomic sequencing. We then demonstrated the method on Prochlorococcus and Synechococcus field populations from the Sargasso Sea and California Current isolated by flow cytometric sorting and found clear environmentally related differences in ecotype distributions and gene abundances. In addition, we saw a significant correspondence between metagenomic libraries sequenced with our technique and regular sequencing of bulk DNA. Our results show that this targeted method is a viable replacement for regular metagenomic approaches and will be useful for identifying the biogeography and genome content of specific marine cyanobacterial populations.

Peer Review #2 of "A localized PCR inhibitor in a porcelain crab suggests a protective role (v0.1)"

“…1) showed the best amplification of fish DNA in reactions using this amount (see below). This is also consistent with other studies using 2 ng of DNA per 20-25 µl PCR reaction, although amplification can be observed at much lower concentrations (Andrade et al 2012;Jin et al, 2012;Bernal-Martínez et al, 2013;Batmalle et al, 2014;Okeke et al, 2014) To assess the presence and distribution of the inhibitor various PCR-based experiments were designed. All PCR reactions were developed on 1.0-1.7% agarose gels stained with ethidium bromide and the intensity of the bands obtained was observed under UV light in an ImageQuant 300 (General Electric) gel imaging system.…”

Techniques for Quantifying Phytoplankton Biodiversity

“…Data are from Martiny et al (2009a). cells from regions with low nutrient concentrations contain a suite of genes for the uptake of nitrogen or phosphorus compounds, but those genes are generally absent in regions with elevated nutrient availability (Martiny et al 2009a,b). This pattern has been observed in Prochlorococcus and Synechococcus as well as multiple heterotrophic bacteria and thus may be a general pattern for marine bacteria (Batmalle et al 2014;Martiny et al 2009a,b;Newton et al 2010); by contrast, this pattern has not been detected in eukaryotic phytoplankton.…”

“…A variant of metagenomics utilizes flow cytometry-based cell sorting to target specific populations (Batmalle et al 2014, Palenik et al 2009, Zehr et al 2008. However, owing to practical limitations in the number of cells sorted, this approach leads to vanishingly small amounts of the DNA.…”

“…However, owing to practical limitations in the number of cells sorted, this approach leads to vanishingly small amounts of the DNA. Thus, the sequencing of sorted populations requires DNA amplification, which can be achieved by either multiple displacement amplification (MDA) or a transposon-based technique (e.g., Nextera) (Batmalle et al 2014). It has been well established that MDA leads to an extensive bias whereby the representations of some genomic regions are highly skewed.…”

Nitrogen assimilation in picocyanobacteria inhabiting the oxygen‐deficient waters of the eastern tropical North and South Pacific

“…Initially, Prochlorococcus was considered unable to use nitrate (), because the original cultured isolates could not use it (Moore et al ). Nevertheless, more recent studies have demonstrated genomic (Martiny et al ; Batmalle et al ; Berube et al ) and indirect physiological (Casey et al ) evidence of assimilation, in both cultured Prochlorococcus and natural assemblages of Prochlorococcus . Analyzing the genomes of different strains of Prochlorococcus , Berube et al () showed that the genomic configuration of the genes related to the assimilation differed among Prochlorococcus strains.…”