The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing

International audienceCurrent sampling of genomic sequence data from eukaryotes is relatively poor, biased, and inadequate to address important questions about their biology, evolution, and ecology; this Community Page describes a resource of 700 transcriptomes from marine microbial eukaryotes to help understand their role in the world's oceans

Deep Panning: Steps towards Probing the IgOme

Background: Polyclonal serum consists of vast collections of antibodies, products of differentiated B-cells. The spectrum of antibody specificities is dynamic and varies with age, physiology, and exposure to pathological insults. The complete repertoire of antibody specificities in blood, the IgOme, is therefore an extraordinarily rich source of information–a molecular record of previous encounters as well as a status report of current immune activity. The ability to profile antibody specificities of polyclonal serum at exceptionally high resolution has been an important and serious challenge which can now be overcome. Methodology/Principal Findings: Here we illustrate the application of Deep Panning, a method that combines the flexibility of combinatorial phage display of random peptides with the power of high-throughput deep sequencing. Deep Panning is first applied to evaluate the quality and diversity of naïve random peptide libraries. The production of very large data sets, hundreds of thousands of peptides, has revealed unexpected properties of combinatorial random peptide libraries and indicates correctives to ensure the quality of the libraries generated. Next, Deep Panning is used to analyze a model monoclonal antibody in addition to allowing one to follow the dynamics of biopanning and peptide selection. Finally Deep Panning is applied to profile polyclonal sera derived from HIV infected individuals. Conclusions/Significance: The ability to generate and characterize hundreds of thousands of affinity-selected peptide

Pie charts depicting the proportion of unique peptides in phage display libraries.

<p>A total of 155,241 inserts were read for the random phage display peptide library (<b>A</b>). 24% of the peptides contained at least one UAA or UGA stop codon (red plus dark red). 58% of the peptides were unique containing a UAG stop codon (light green) of these some exist in multiple copies (3% of the total, dark green). 15% of the peptides were completely devoid of stop codons (blue, less than 1% had 2–5 copies). Pie Chart (<b>B</b>) depicts the same set of peptides devoid of all those that had detectable frameshifted inserts (37,223 inserts leaving 118,018 functional peptides of which ca 1% contained stop codons UAA and UGA nonetheless). A second library was constructed in DH5alpha <i>supE144</i> cells (<b>C</b>). Values below 1% are not given.</p

Deep Panning with mAb GV4H3.

<p>(<b>A</b>) Mapitope prediction of the GV4H3 epitope on HIV gp120. The top 20 peptides of Capture #2 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041469#pone-0041469-t001" target="_blank">Table 1</a>) were used as the dataset for Mapitope prediction of the GV4H3 epitope. The single predicted cluster comprises two discontinuous segments of the antigen (green and blue) brought to flank the core of the epitope (residues 221–226, pink). (<b>B-E</b>) MEME analysis of the GV4H3 derived peptides. The 20 top most frequent peptides of Capture #3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041469#pone-0041469-t001" target="_blank">Table 1</a>) generated a major motif “AGWAV”. This motif (<b>B</b>) and three additional motifs are identified when all 4,823 peptides are analyzed. The “VGF” motif (<b>C</b>) is a simpler version of the major motif. The two additional minor motifs (<b>D</b> and <b>E</b>) do not have obvious similarity to the epitope of the mAb. The “ADGIGGG” motif clearly corresponds with the most frequent peptide ADGIVGW (see text). The numbers in red represent the number of unique peptides that define each motif.</p

BLASTP analysis of HIVIG-captured peptides against viral coat proteins.

<p>Of the 223 top unique HIVIG-captured peptides, 18 (8%) scored hits in BLASTP analysis against the HIV-1_HXB2 gp160 (blue). Repeating this procedure with the same protein but scrambled gives an average value of 5.5 hits when performed 1,000 times (2.5%±2.2 s.d., red). The differences between native and scrambled coat proteins BLASTP results of 11 other RNA viruses were not found to be significant. <i>*P</i><0.01.</p

Three rounds of panning with mAb GV4H3.

<p>GV4H3 mAb was used to bio-pan the 7 mer random peptide library 3 consecutive rounds of panning (Capture #1 through #3) and compared with the naïve library. For each sample the 20 top most frequent peptides are given along with the number of times they appear. The number of unique versus total peptides is shown as well. Numbers in parentheses represent the percent value of the total peptides for each category. Bold sequences indicate peptides that are carried over from Capture #1. Bold and Italic sequences indicate peptides carried over from Capture #2.</p

The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing.

Current sampling of genomic sequence data from eukaryotes is relatively poor, biased, and inadequate to address important questions about their biology, evolution, and ecology; this Community Page describes a resource of 700 transcriptomes from marine microbial eukaryotes to help understand their role in the world's oceans

A schematic of the major lineages in the eukaryotic tree of life, showing the relationships between lineages for which genomic resources are currently available and those that have been targeted by the MMETSP.

<p>Lineages with complete genomes according to the GOLD database, as summarized in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001889#pbio.1001889-Burki1" target="_blank">[3]</a>, are indicated by a solid line leading to that group, whereas lineages with no complete genome are represented by a dashed line. Lineages where at least one MMETSP transcriptome is complete or underway are indicated with a red dot by the name. Major lineages discussed in the text have been named and color-coded, but for clarity, some major lineages have not been labeled.</p

Comparing the diversity of microbial eukaryotes at one marine site with that represented in genome data and the MMETSP project.

<p>(A) Taxon assignments for 930 Small Subunit (SSU) rRNA gene sequences from environmental clone libraries built using DNA from three size fractions in sunlit surface waters of the North Pacific Ocean. Four hundred and five sequences corresponding to Syndiniales (nonphotosynthetic members of the dinoflagellate lineage, often referred to as MALV1 and MALV2) were excluded for visualization purposes. Syndiniales are not represented in any complete genome data or the MMETSP, and the vast majority are only known as sequences from uncultivated taxa that often dominate clone libraries <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001889#pbio.1001889-Massana1" target="_blank">[22]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001889#pbio.1001889-Massana2" target="_blank">[31]</a>. Filter size fractions were 0.1 to <0.8 µm, 0.8 to <3 µm, and 3 to <20 µm. This graph is only intended to give a snapshot of one marine sample; relative distributions vary based on distance from shore and depth, and several studies provide more detailed reviews of available SSU rRNA gene sequence surveys, see e.g., <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001889#pbio.1001889-Amin1" target="_blank">[21]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001889#pbio.1001889-Not1" target="_blank">[32]</a>. (B) Taxonomic diversity of eukaryotes with complete genome sequences, as summarized in the Genomes Online Database (GOLD: <a href="http://genomesonline.org" target="_blank">http://genomesonline.org</a>). Note that multicellular organisms are included (unlike in A or C); animals, land plants, and multicellular rhodophytes are included in the opisthokont, viridiplantae, and rhodophyte categories, respectively. (C) Taxon breakdown of the MMETSP sequencing project, collapsed at the strain level (for some strains, cells were grown under multiple conditions and these have been counted only once). (D) Comparison of currently available complete genomes and MMETSP transcriptomes by Class for two diverse and well-studied groups of algae, prasinophytes <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001889#pbio.1001889-Marin1" target="_blank">[14]</a> and dinoflagellates <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001889#pbio.1001889-Fensome1" target="_blank">[15]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001889#pbio.1001889-Saldarriagaa1" target="_blank">[16]</a>. For both lineages, genomes are broken down by Class on the left and MMETSP transcriptomes on the right.</p