Single cell genomics yields a wide diversity of small planktonic protists across major ocean ecosystems.

Marine planktonic protists are critical components of ocean ecosystems and are highly diverse. Molecular sequencing methods are being used to describe this diversity and reveal new associations and metabolisms that are important to how these ecosystems function. We describe here the use of the single cell genomics approach to sample and interrogate the diversity of the smaller (pico- and nano-sized) protists from a range of oceanic samples. We created over 900 single amplified genomes (SAGs) from 8 Tara Ocean samples across the Indian Ocean and the Mediterranean Sea. We show that flow cytometric sorting of single cells effectively distinguishes plastidic and aplastidic cell types that agree with our understanding of protist phylogeny. Yields of genomic DNA with PCR-identifiable 18S rRNA gene sequence from single cells was low (15% of aplastidic cell sorts, and 7% of plastidic sorts) and tests with alternate primers and comparisons to metabarcoding did not reveal phylogenetic bias in the major protist groups. There was little evidence of significant bias against or in favor of any phylogenetic group expected or known to be present. The four open ocean stations in the Indian Ocean had similar communities, despite ranging from 14°N to 20°S latitude, and they differed from the Mediterranean station. Single cell genomics of protists suggests that the taxonomic diversity of the dominant taxa found in only several hundreds of microliters of surface seawater is similar to that found in molecular surveys where liters of sample are filtered.

The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403.

Lactococcus lactis is a nonpathogenic AT-rich gram-positive bacterium closely related to the genus Streptococcus and is the most commonly used cheese starter. It is also the best-characterized lactic acid bacterium. We sequenced the genome of the laboratory strain IL1403, using a novel two-step strategy that comprises diagnostic sequencing of the entire genome and a shotgun polishing step. The genome contains 2,365,589 base pairs and encodes 2310 proteins, including 293 protein-coding genes belonging to six prophages and 43 insertion sequence (IS) elements. Nonrandom distribution of IS elements indicates that the chromosome of the sequenced strain may be a product of recent recombination between two closely related genomes. A complete set of late competence genes is present, indicating the ability of L. lactis to undergo DNA transformation. Genomic sequence revealed new possibilities for fermentation pathways and for aerobic respiration. It also indicated a horizontal transfer of genetic information from Lactococcus to gram-negative enteric bacteria of Salmonella-Escherichia group.

Human-ovine comparative sequencing of a 250-kb imprinted domain encompassing the callipyge (clpg) locus and identification of six imprinted transcripts: DLK1, DAT, GTL2, PEG11, antiPEG11, and MEG8.

Two ovine BAC clones and a connecting long-range PCR product, jointly spanning approximately 250 kb and representing most of the MULGE5-OY3 marker interval known to contain the clpg locus, were completely sequenced. The resulting genomic sequence was aligned with its human ortholog and extensively annotated. Six transcripts, four of which were novel, were predicted to originate from within the analyzed region and their existence confirmed experimentally: DLK1, DAT, GTL2, PEG11, antiPEG11, and MEG8. RT-PCR experiments performed on a range of tissues sampled from an 8-wk-old animal demonstrated the preferential expression of all six transcripts in skeletal muscle, which suggests that they are under control of common regulatory elements. The six transcripts were also shown to be subject to parental imprinting: DLK1, DAT, and PEG11 were shown to be paternally expressed and GTL2, antiPEG11, and MEG8 to be maternally expressed.

Characterization and repeat analysis of the compact genome of the freshwater pufferfish Tetraodon nigroviridis.

Tetraodon nigroviridis is a freshwater pufferfish 20-30 million years distant from Fugu rubripes. The genome of both tetraodontiforms is compact, mostly because intergenic and intronic sequences are reduced in size compared to other vertebrate genomes. The previously uncharacterized Tetraodon genome is described here together with a detailed analysis of its repeat content and organization. We report the sequencing of 46 megabases of bacterial artificial chromosome (BAC) end sequences, which represents a random DNA sample equivalent to 13% of the genome. The sequence and location of rRNA gene clusters, centromeric and subtelocentric satellite sequences have been determined. Minisatellites and microsatellites have been cataloged and notable differences were observed in comparison with microsatellites from Fugu. The genome contains homologies to all known families of transposable elements, including Ty3-gypsy, Ty1-copia, Line retrotransposons, DNA transposons, and retroviruses, although their overall abundance is <1%. This structural analysis is an important prerequisite to sequencing the Tetraodon genome.

Estimate of human gene number provided by genome-wide analysis using Tetraodon nigroviridis DNA sequence.

The number of genes in the human genome is unknown, with estimates ranging from 50,000 to 90,000 (refs 1, 2), and to more than 140,000 according to unpublished sources. We have developed 'Exofish', a procedure based on homology searches, to identify human genes quickly and reliably. This method relies on the sequence of another vertebrate, the pufferfish Tetraodon nigroviridis, to detect conserved sequences with a very low background. Similar to Fugu rubripes, a marine pufferfish proposed by Brenner et al. as a model for genomic studies, T. nigroviridis is a more practical alternative with a genome also eight times more compact than that of human. Many comparisons have been made between F. rubripes and human DNA that demonstrate the potential of comparative genomics using the pufferfish genome. Application of Exofish to the December version of the working draft sequence of the human genome and to Unigene showed that the human genome contains 28,000-34,000 genes, and that Unigene contains less than 40% of the protein-coding fraction of the human genome.

Karyotype and chromosome location of characteristic tandem repeats in the pufferfish Tetraodon nigroviridis.

Karyotype analysis of Tetraodon nigroviridis, a pufferfish of the family Tetraodontidae with a small compact genome (385 Mb) which is currently being investigated in our laboratory, indicates that this species has 2n = 42 chromosomes. The small chromosome size (the largest pair measuring less than 3 microm) has complicated accurate chromosome pairing based on morphology alone. DAPI staining, however, provides a banding-like pattern. Because of quantitative variations of some heterochromatin classes, the chromosome formula can not be established precisely, but is estimated to include approximately 20 meta- or submetacentric chromosomes and 22 subtelocentric chromosomes. A centromeric satellite, telomeric repeats, and the major and minor rRNA clusters have been localized unequivocally by FISH. As a result, the 28S and 5S rDNA sequences can be used as chromosome-specific probes.

The gene encoding the mouse homologue of the human osteoclast-specific 116-kDa V-ATPase subunit bears a deletion in osteosclerotic (oc/oc) mutants.

Osteosclerosis (oc) is an autosomal recessive lethal mutation that impairs bone resorption by osteoclasts, and induces a general increase of bone density in affected mice. Genetic mapping of the oc mutation was used as a backbone in a positional cloning approach in the pericentromeric region of mouse chromosome 19. Perfect cosegregation of the osteopetrotic phenotype with polymorphic markers enabled the construction of a sequence-ready bacterial artificial chromosome (BAC) contig of this region. Genomic sequencing of a 200-kb area revealed the presence of the mouse homologue to the human gene encoding the osteoclast-specific 116-kDa subunit of the vacuolar proton pump. This gene was located recently on human 11q13, a genomic region conserved with proximal mouse chromosome 19. Sequencing of the 5' end of the gene in oc/oc mice showed a 1.6-kb deletion, including the translation start site, which impairs genuine transcription of this subunit. The inactivation of this osteoclast-specific vacuolar proton ATPase subunit could be responsible for the lack of this enzyme in the apical membranes of osteoclast cells in oc/oc mice, thereby preventing the resorption function of these cells, which leads to the osteopetrotic phenotype.

Sequence analysis of the NS5A protein of European hepatitis C virus 1b isolates and relation to interferon sensitivity.

Japanese studies have defined the discrete 2209-2248 amino acid region of the non-structural 5A protein (NS5A(2209-2248)) of hepatitis C virus genotype 1b (HCV 1b) isolates as the interferon sensitivity determining region (ISDR). European studies did not confirm these results since most of the ISDR sequences harboured an intermediate profile. Recently, a direct interaction between the NS5A protein, involving the ISDR, and the interferon-induced protein kinase (PKR) has been reported and presented as a possible explanation of HCV interferon resistance. In the present study, the entire NS5A amino acid sequence from 11 resistant and eight sensitive strains from European HCV 1b isolates was inferred from direct sequencing. The previously described important amino acid stretches and positions in NS5A were compared between the resistant and sensitive groups. Although some variations were observed, no clear differences could be directly correlated with the interferon sensitivity. However, sensitive strains were different, owing to more amino acid changes when compared to a consensus sequence from all strains. The carboxy-terminal region and especially the previously reported NS5A/V3 region showed most of the variations. Moreover, the conformational analysis of NS5A by secondary structure prediction allowed the differentiation of most sensitive strains from resistant ones. It was concluded that other regions different from ISDR were involved in resistance to interferon maybe via the interaction between NS5A and PKR.

Amplification and detection of the terminal 3' non-coding region of hepatitis C virus isolates.

A reverse transcription polymerase chain reaction (RT-PCR) assay was set up to amplify, from chronically infected patients, the recently discovered hepatitis C virus (HCV) 3'non-coding region (3'NCR). A panel of 149 samples was tested by RT-PCR for the 3'NCR. Two detection methods of amplified products were evaluated: ethidium bromide staining on 3% agarose gel electrophoresis and DNA enzyme immunoassay ("DEIA"). Results were compared with those obtained by amplification of the 5' non-coding region (5'NCR), i.e. the "Amplicor" HCV RNA qualitative assay. Genotype distribution of the 86 Amplicor-positive samples was subtype 1a: n = 15 (17.4%); subtype 1b: n = 32 (37.2%); subtype 2a/2c: n = 7 (8.1%); type 3: n = 25 (29%); type 4: n = 2 (2.3%); type 5: n = 1 (1.2%); not determined: n = 4 (2.3%). Sixty-three sera were HCV RNA-Amplicor-negative, 32 of which were from HCV-seronegative patients and 31 from HCV-seropositive patients. All seronegative samples were negative by both PCR methods. None of the Amplicor-negative samples from seropositive patients were positive by the 3'NCR assay. Forty-seven (54.7%) and 83 (96.5%) of the 86 Amplicor-HCV-RNA-positive samples were positive after ethidium bromide staining and by the 3'NCR assay using DEIA, respectively. The limit of detection by end-point dilution was lower with Amplicor. No difference between genotypes was detected for the 3'NCR RT-PCR, and a high degree of concordance was obtained between the Amplicor and the 3'NCR DEIA results (97.4%). Nevertheless, further studies are needed before the 3'NCR RT-PCR assay could be used instead of the 5'NCR RT-PCR for diagnostic purposes.