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. 2014 Sep 30;1:140034. doi: 10.1038/sdata.2014.34

Genomes of diverse isolates of the marine cyanobacterium Prochlorococcus

Steven J Biller 1,a, Paul M Berube 1, Jessie W Berta-Thompson 1,2, Libusha Kelly 1,†, Sara E Roggensack 1, Lana Awad 1, Kathryn H Roache-Johnson 3, Huiming Ding 1,4, Stephen J Giovannoni 5, Gabrielle Rocap 6, Lisa R Moore 3, Sallie W Chisholm 1,4,b
PMCID: PMC4421930  PMID: 25977791

Abstract

The marine cyanobacterium Prochlorococcus is the numerically dominant photosynthetic organism in the oligotrophic oceans, and a model system in marine microbial ecology. Here we report 27 new whole genome sequences (2 complete and closed; 25 of draft quality) of cultured isolates, representing five major phylogenetic clades of Prochlorococcus. The sequenced strains were isolated from diverse regions of the oceans, facilitating studies of the drivers of microbial diversity—both in the lab and in the field. To improve the utility of these genomes for comparative genomics, we also define pre-computed clusters of orthologous groups of proteins (COGs), indicating how genes are distributed among these and other publicly available Prochlorococcus genomes. These data represent a significant expansion of Prochlorococcus reference genomes that are useful for numerous applications in microbial ecology, evolution and oceanography.

Background & Summary

As the smallest (<1 μm diameter) and most abundant (3×1027 cells) photosynthetic organism on the planet1, Prochlorococcus has a unique status in the microbial world. This unicellular marine cyanobacterium is found throughout the euphotic zone of the open ocean between ~45°N and 40°S, where it carries out a notable fraction of global photosynthesis1, 2. The group, which would be considered a single microbial ‘species’ by the traditional measure of >97% 16S rRNA similarity, is composed of multiple phylogenetically distinct clades (Figure 1) (as defined by either rRNA internal transcribed spacer (ITS)3 or whole-genome sequences4) which are physiologically distinct. Adaptations for optimal growth at different light intensities differentiate deeply branching groups of Prochlorococcus into high light (HL) and low light (LL) adapted clades3,5–8.

Figure 1.

Figure 1

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Prochlorococcus strains sequenced in this work. ITS-based phylogeny of the strains included in this data set (names in bold, with *) in relation to previously sequenced Prochlorococcus. Phylogenetic clade affiliation4,6 is indicated at right; closed circles indicate nodes with bootstrap support >75%. HL—High light adapted; LL—Low light adapted, as determined by physiological studies of some of the isolates3,5,7.

Prochlorococcus have the smallest genomes of any known free-living photosynthetic cell, ranging from ~1.6 to 2.7 Mbp4. While they all share a core set of genes present in all strains, there exists remarkable diversity in gene content among isolates. The group has an ‘open’ pan-genome, i.e. each newly sequenced genome typically contains many new genes never before seen in Prochlorococcus 4. Given the abundance of Prochlorococcus, studies of their genomic and metagenomic features have provided numerous insights into features of ocean ecosystems9–17. In addition, the group has proven to be a valuable system for studying microbial evolution18,19, genome streamlining20,21, and the relationship between genotypic, phenotypic and ecological variation in marine populations3,7,22. Since Prochlorococcus is abundant in surface waters, these reference genomes have also been extremely valuable for interpreting marine metagenomic and metatranscriptomic datasets14,23–28.

To advance our understanding of Prochlorococcus genetic diversity, we sequenced the genomes of 27 Prochlorococcus strains from a variety of ocean environments. The strains sequenced included both previously reported strains as well as eight new isolates (Table 1). The newly isolated strains come from ocean regions that previously only had few or no cultured representatives and substantially expand the number of cultured Prochlorococcus available for five major clades. These results demonstrate the applicability of high-throughput dilution-to-extinction cultivation approaches29 to Prochlorococcus.

Table 1. Origin of the Prochlorococcus strains sequenced in this study.

Strain Alternate Name Ecotype/Clade 4,57 Isolation location Isolation (Lat/Lon) Isolation depth (m) Isolation date Strain reference
EQPAC1 RCC278 eMED4/HLI Equatorial Pacific 0°N 180°W 30 Roscoff Culture Collection
GP2 eMIT9312/HLII Western Pacific 8°N 136°E 150 Sep-1992 32
MIT0604 eMIT9312/HLII Station ALOHA/North Pacific 22.75°N 158°W 175 May-2006 This work
MIT9107 eMIT9312/HLII Tropical Pacific 15°S 135°W 25 8-Aug-1991 33
MIT9116 eMIT9312/HLII Tropical Pacific 15°S 135°W 25 8-Aug-1991 6
MIT9123 eMIT9312/HLII Tropical Pacific 15°S 135°W 25 8-Aug-1991 6
MIT9201 eMIT9312/HLII Tropical Pacific 12°S 145.42°W Surface 26-Sep-1992 5
MIT9302 eMIT9312/HLII Sargasso Sea 34.76°N 66.19°W 100 15-Jul-1993 3
MIT9311 eMIT9312/HLII Gulf stream 37.51°N 64.24°W 135 17-Jul-1993 6
MIT9314 eMIT9312/HLII Gulf stream 37.51°N 64.24°W 180 17-Jul-1993 6
MIT9321 eMIT9312/HLII Equatorial Pacific 1°N 92°W 50 12-Nov-1993 6
MIT9322 eMIT9312/HLII Equatorial Pacific 0.27°N 93°W Surface 16-Nov-1993 6
MIT9401 eMIT9312/HLII Sargasso Sea 35.5°N 70.4°W Surface May-1994 6
SB eMIT9312/HLII Western Pacific 35°N 138.3°E 40 1-Oct-1992 32
MIT0801 HTCC 1603 eNATL/LLI BATS/Sargasso Sea 31.67°N 64.17°W 40 25-Mar-2008 This work
PAC1 eNATL/LLI Station ALOHA/North Pacific 22.75°N 158°W 100 1992 34,35
LG eSS120/LLII,III Sargasso Sea 28.98°N 64.35°W 120 30-May-1988 36
MIT0601 eMIT9211/LLII,III Station ALOHA/North Pacific 22.75°N 158°W 125 17-Nov-2006 This work
MIT0602 eSS120/LLII,III Station ALOHA/North Pacific 22.75°N 158°W 125 17-Nov-2006 This work
MIT0603 eSS120/LLII,III Station ALOHA/North Pacific 22.75°N 158°W 125 17-Nov-2006 This work
SS2 eSS120/LLII,III Sargasso Sea 28.98°N 64.35°W 120 30-May-1988 6
SS35 eSS120/LLII,III Sargasso Sea 28.98°N 64.35°W 120 30-May-1988 6
SS51 eSS120/LLII,III Sargasso Sea 28.98°N 64.35°W 120 30-May-1988 6
SS52 eSS120/LLII,III Sargasso Sea 28.98°N 64.35°W 120 30-May-1988 6
MIT0701 HTCC 1600 eMIT9313/LLIV South Atlantic 13.45°S 0.04°W 150 1-Dec-2007 This work
MIT0702 HTCC 1601 eMIT9313/LLIV South Atlantic 13.45°S 0.04°W 150 1-Dec-2007 This work
MIT0703 HTCC 1602 eMIT9313/LLIV South Atlantic 13.45°S 0.04°W 150 1-Dec-2007 This work
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The genome sequences reported here represent a notable increase in the number of genome sequences available from the major phylogenetic clades with existing cultured representatives. While many genomes differed greatly in gene content, other sets are very closely related and differ primarily by single nucleotide polymorphisms (e.g., LG, SS2, SS35, SS51, SS52, SS120; and MIT0701, MIT0702, and MIT0703). Thus, this dataset encompasses a broad range of pairwise genomic diversity among Prochlorococcus strains.

Most genomes were sequenced to draft status; two were closed (Table 2). We used two annotation methods to identify the potential functions of genes in the genomes. Genes were first called and annotated by the RAST pipeline30. To expand on these predictions—especially for the myriad genes of unknown function—we also derived annotations from an independent pipeline, Argot231. To facilitate the utility of these genomes for comparative genomics and evolutionary studies, we define a set of pre-computed orthologous gene clusters for Prochlorococcus. All cluster data are supplied in this data set (Data Citation 1 and Data Citation 2).

Table 2. Genome characteristics and assembly statistics.

Strain Clade 4 Assembly size (bp) %GC No. contigs N50 (bp) No. coding sequences NCBI accession*
*For the Whole Genome Shotgun projects deposited at DDBJ/EMBL/GenBank: the version described in this paper is version JN**01000000.              
EQPAC1 HLI 1,654,739 30.8 8 328,627 1,954 JNAG00000000
GP2 HLII 1,624,310 31.2 11 416,038 1,884 JNAH00000000
MIT0604 HLII 1,780,061 31.2 1 1,780,061 2,085 CP007753
MIT9107 HLII 1,699,937 31.0 13 170,362 1,991 JNAI00000000
MIT9116 HLII 1,685,398 31.0 22 117,620 1,972 JNAJ00000000
MIT9123 HLII 1,697,748 31.0 18 137,374 2,005 JNAK00000000
MIT9201 HLII 1,672,416 31.3 21 145,955 1,989 JNAL00000000
MIT9302 HLII 1,745,343 31.1 17 242,124 2,015 JNAM00000000
MIT9311 HLII 1,711,064 31.2 17 189,094 1,983 JNAN00000000
MIT9314 HLII 1,690,556 31.2 16 221,824 1,990 JNAO00000000
MIT9321 HLII 1,658,664 31.2 10 259,210 1,956 JNAP00000000
MIT9322 HLII 1,657,550 31.2 11 367,597 1,959 JNAQ00000000
MIT9401 HLII 1,666,808 31.2 17 110,519 1,972 JNAR00000000
SB HLII 1,669,823 31.5 4 1,237,529 1,933 JNAS00000000
MIT0801 LLI 1,929,203 34.9 1 1,929,203 2,287 CP007754
PAC1 LLI 1,841,163 35.1 20 182,484 2,264 JNAX00000000
LG LLII,III 1,754,063 36.4 14 326,623 1,973 JNAT00000000
MIT0601 LLII,III 1,707,342 37.0 6 547,047 1,934 JNAU00000000
MIT0602 LLII,III 1,750,918 36.3 9 511,704 1,998 JNAV00000000
MIT0603 LLII,III 1,752,482 36.3 7 434,668 2,015 JNAW00000000
SS2 LLII,III 1,752,772 36.4 19 187,268 1,989 JNAY00000000
SS35 LLII,III 1,751,015 36.4 9 446,270 1,977 JNAZ00000000
SS51 LLII,III 1,746,977 36.4 12 232,789 1,974 JNBD00000000
SS52 LLII,III 1,754,053 36.4 22 124,224 1,987 JNBE00000000
MIT0701 LLIV 2,592,571 50.6 53 84,463 3,079 JNBA00000000
MIT0702 LLIV 2,583,057 50.6 61 76,101 3,066 JNBB00000000
MIT0703 LLIV 2,575,057 50.6 61 81,186 3,054 JNBC00000000
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These genomes should be useful to researchers interested in many aspects of marine microbial ecology and evolution. Since the genomes are from cultured isolates, hypotheses generated from these data can be tested in laboratory experiments. The genomes will also greatly facilitate the interpretation of transcriptomic and proteomic studies, as well as meta-‘omic’ data from field studies where Prochlorococcus is a dominant phototroph.

Methods

Culturing and strain isolations

Many of the strains sequenced have been previously described3,5,6,32–36 (Table 1); 8 are reported here for the first time. All cultures were unialgal; this was initially determined crudely by flow cytometry profiles, and then more specifically by confirming the presence of only one cyanobacterial 16S rRNA ITS sequence in the culture. All cultures except SB and MIT0604 contained heterotrophic bacteria. Cultures were maintained in acid-washed glassware in Pro99 media37 prepared with 0.2 μm filtered, autoclaved seawater collected from Vineyard Sound, MA or the Sargasso Sea under either a 14:10 light:dark cycle at 24 °C or constant light flux at 21 °C. Light levels were 30–40 μmol Q m−2 s−1 for high-light adapted strains, and 10–20 μmol Q m−2 s−1 for low-light adapted strains.

MIT0601, MIT0602, MIT0603, and MIT0604 were derived from enrichment cultures initiated with seawater obtained from the North Pacific Ocean at Station ALOHA (22.75°N, 158°W) on Hawai’i Ocean Time-series (HOT) cruise 181. The seawater was amended with nitrogen, phosphorous and trace metals (PRO2 nutrient additions37, except all nitrogen sources were replaced by 0.217 mM sodium nitrate).

Strains MIT0701, MIT0702, and MIT0703 were isolated from the South Atlantic (CoFeMUG cruise KN192-05, station 13, 13.45 °S, 0.04 °W) at 150 m using a high throughput culturing method29 adapted for phototrophs. The seawater used for isolations was first filtered through a 1 μm filter with no amendments and kept in the dark at 18–20 °C for 21 days. The total red fluorescing phytoplankton population (1×105 cells ml−1 determined with a Guava EasyCyte flow cytometer) was diluted in PRO3V media37 made with the same South Atlantic water that had been filtered through a 0.1 μm Supor 142 mm filter, then autoclaved to sterilize. This media contained 100 μM NH4Cl, 10 μM NaH2PO4, PRO2 trace metals37 and f/2 vitamins (0.1 μg l−1 cyanocobalamin, 20 g l−1 thiamin and 1 μg l−1 biotin38,39). Ten cells were dispensed into 1 ml volumes in a 48-well polystyrene multiwell culture plate and incubated at 20 °C in ~20 μmol Q m−2 s−1 (14:10 light:dark) for 2 months.

MIT0801 was isolated in a similar manner, but from seawater obtained from 40 m depth at the Bermuda Atlantic Time-series station (BATS; 31.67 °N, 64.16 °W) that had been sitting in the dark for 5 days. The same PRO3V media recipe was made with 0.1 μm filtered and autoclaved BATS seawater, and 2.5 cells (on average) were dispensed in 5 ml volume in Teflon plates (prepared as described29). Cells were detected within 1 month of enrichment.

DNA sequencing and assembly

Genomes were sequenced from genomic DNA collected from 20 ml laboratory cultures. Cells were collected by centrifugation (10,000g, 10 min), the pellet transferred into a 2 ml tube and frozen at −80 °C. Genomic DNA was isolated using the QIAamp DNA mini kit (Qiagen). 2 μg of DNA was then used to construct an Illumina sequencing library as previously described40, except that the bead:sample ratios in the double solid phase reversible immobilization (dSPRI) size-selection step were 0.7 followed by 0.15, resulting in fragments with an average size of ~340 bp (range: 200–600 bp). PAC1 and EQPAC1 libraries were constructed using dSPRI bead:sample ratios of 0.9 followed by 0.21, yielding an average size of ~220 bp. DNA libraries were sequenced on an Illumina GAIIx, producing 200+200 nt paired reads, at the MIT BioMicro Center. An average of 1.6 million paired-end reads were obtained for each genome.

Low quality regions of sequencing data were removed from the raw Illumina data using quality_trim (V3.2, from the CLC Assembly Cell package; CLC bio) with default settings (at least 50% of the read must be of a minimum quality of 20). Paired-end reads were overlapped using the SHE-RA algorithm41, keeping any resulting overlapping sequences with an overlap score >0.5. For all genomes except PAC1 and EQPAC1, the overlapped reads, as well as the trimmed paired-end reads that did not overlap, were assembled using the Newbler assembler (V2.6; 454/Roche) with the following parameters: ‘-e 200 –rip.’ Contigs <1 Kbp were discarded at this stage.

Reads for PAC1 and EQPAC1 were assembled using clc_novo_assemble (V3.2, from the CLC Assembly Cell package; CLC bio) with a minimum contig length of 500 bp and automatic wordsize determination enabled. These initial contigs were searched against a custom database of marine microbial genomes9 using BLAST42 to identify contigs with a closest match to Prochlorococcus. Sequencing reads belonging to the putative Prochlorococcus contigs were then identified by mapping the raw sequences to these contigs using clc_ref_asssemble_long (CLC bio). The Prochlorococcus-like reads were then re-assembled using clc_novo_assemble using the same parameters as above to produce the final assembly, now largely free of heterotrophic sequences.

MIT0604 and MIT0801 were completed to finished quality with no gaps by directed PCR reactions to sequence contig junctions, combined with Pacific Biosciences long sequencing reads. Contigs were ordered into putative scaffolds based on their similarity to closely related closed Prochlorococcus genomes, as determined by Mauve43. PCR primers specific to the ends of putatively adjacent contigs were designed and used to amplify the junctions between contigs. Purified PCR products were sequenced by Sanger chemistry at the MGH DNA core facility, and the resulting sequences used to join contigs in Consed44. This resulted in a highly improved but still incomplete assembly. To span difficult repeat regions in MIT0801, we obtained long Pacific Biosciences sequences. We obtained DNA from 25 ml cultures using the Epicentre Masterpure kit (Epicentre) and sequenced this at the Yale Center for Genome Analysis. We combined this set of long but low quality reads with the high quality Illumina short reads obtained previously using the PacBioToCA software45, to produce assemblies with a reduced number of contigs. These contigs were aligned to the PCR-improved contigs described above, and the final gaps were closed with a small number of additional directed PCR reactions (as described above) using the Geneious sequence analysis package (V6.1, Biomatters), until the genomes were closed.

As most of the Prochlorococcus cultures sequenced were known to contain heterotrophs, we identified the most ‘Prochlorococcus-like’ contigs from non-axenic cultures by searching each resulting contig against a custom database of sequenced marine microbial genomes9 using BLAST42. Contigs with a best match to a non-Prochlorococcus genome were removed from the assembly. Subsequent examination of these contig sets indicated that a number of shorter sequences (generally <10 kbp) with significant heterotroph-like stretches had passed through the initial filtering steps. To remove these questionable contigs from the assemblies, we manually examined each <10 kbp contig using the RAST annotation server (see below), and only kept those contigs with clear homology to previously sequenced and closed Prochlorococcus or Synechococcus genomes. Although these filtering steps may have removed a small amount of true Prochlorococcus sequence from the final assembly, we considered missing a few genes preferable to misrepresenting heterotroph sequences as Prochlorococcus.

Examination of the non-cyanobacterial 16S rRNA genes found within these data indicate that the most abundant heterotrophs in the cultures were members of the Alteromonadales, Flavobacteriales, Rhodospirillales, Halomonadaceae, and Sphingobacteriales. We have included a separate data file containing all of the assembled contigs—including those from co-cultured heterotrophs—for anyone who is interested (Data File 4).

Genome annotation

The assembled contigs for each genome were annotated using the RAST server30 against FIGfam release 49. Additional functional annotation for all genes called by RAST were generated by the Argot2 server31, using default settings.

To confirm the rRNA-based phylogeny of these strains, rRNA ITS sequences were aligned in ARB46 and maximum likelihood phylogenies calculated in PhyML version 2012041247, using the HKY85 model of nucleotide substitution, a fixed proportion of invariable sites, and non-parametric bootstrap analysis with 100 replicates.

Clusters of orthologous groups of proteins (COGs) were computed, as described elsewhere48, on a data set comprised of previously sequenced Prochlorococcus and Synechococcus strains4,10,16,17,49–53, the new Prochlorococcus genomes described here, 11 Prochlorococcus single-cell genomes12 and two consensus metagenomic assemblies14 (Data Citation 1). To facilitate comparisons among genomes, we re-annotated 16 previously sequenced Prochlorococcus genomes (Table 3) with the RAST pipeline as described above; this ensured that a uniform methodology for gene calling and functional annotation was used. Single cell genomes12 were not re-annotated due to difficulties encountered using this pipeline on such fragmented contigs; instead, we utilized the ORFs previously defined in GenBank. Detailed information regarding these updated annotations is provided (Data Citation 1 and Data Citation 2).

Table 3. Previously sequenced Prochlorococcus genomes included in the cyanobacterial clusters of orthologous groups of proteins (CyCOG) definitions.

Name Genome source Clade Assembly size (bp) %GC No. coding sequences* NCBI accession Sequence reference
*For the cultured isolate and metagenomic assembly genomes, this value represents the number of coding sequences as predicted in this study using the RAST pipeline; these values may differ from those previously published for this reason. Re-annotation data is included in this dataset (Data Citation 1 and Data Citation 2).              
MED4 Cultured isolate HLI 1,657,990 30.8 1,959 BX548174 10
MIT9515 Cultured isolate HLI 1,704,176 30.8 1,951 CP000552 4
AS9601 Cultured isolate HLII 1,669,886 31.3 1,944 CP000551 4
MIT9202 Cultured isolate HLII 1,691,453 31.1 2,000 DS999537 49
MIT9215 Cultured isolate HLII 1,738,790 31.1 2,035 CP000825 4
MIT9301 Cultured isolate HLII 1,641,879 31.3 1,925 CP000576 4
MIT9312 Cultured isolate HLII 1,709,204 31.2 1,982 CP000111 16
UH18301 Cultured isolate HLII 1,654,648 31.2 1,947 PRJNA47033 50
W6 Single cell amplified genome HLII 385,307 31.3 646 ALPK00000000 12
HNLC2 Metagenomic assembly HLIII 1,484,494 30.3 1,701 GL947595 14
W3 Single cell amplified genome HLIII 339,045 30.7 529 ALPC00000000 12
W5 Single cell amplified genome HLIII 99,467 29.8 212 ALPL00000000 12
W7 Single cell amplified genome HLIII 905,221 30.7 989 ALPE00000000 12
W8 Single cell amplified genome HLIII 841,756 31.4 917 ALPF00000000 12
W9 Single cell amplified genome HLIII 420,150 30.7 638 ALPG00000000 12
HNLC1 Metagenomic assembly HLIV 1,569,623 29.8 1,830 GL947594 14
W10 Single cell amplified genome HLIV 561,998 30.8 892 ALPH00000000 12
W11 Single cell amplified genome HLIV 766,829 30.6 929 ALPI00000000 12
W12 Single cell amplified genome HLIV 423,437 29.6 602 ALPJ00000000 12
W2 Single cell amplified genome HLIV 1,266,767 30.5 1,374 ALPB00000000 12
W4 Single cell amplified genome HLIV 765,485 29.9 819 ALPD00000000 12
NATL1A Cultured isolate LLI 1,864,731 35.0 2,242 CP000553 4
NATL2A Cultured isolate LLI 1,842,899 35.1 2,194 CP000095 4
MIT9211 Cultured isolate LLII,III 1,688,963 38.0 1,943 CP000878 4
SS120 Cultured isolate LLII,III 1,751,080 36.4 1,973 AE017126 17
MIT9303 Cultured isolate LLIV 2,682,675 50.0 3,253 CP000554 4
MIT9313 Cultured isolate LLIV 2,410,873 50.7 2,993 BX548175 10
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Orthologous gene clusters were defined based on reciprocal best blastp scores (with an e-value cutoff of 1e−5); the sequence alignment length had to be at least 75% of the shorter protein, with at least a 35% identity. Additional orthologous genes that did not pass this criterion were added to clusters based on HMM profiles constructed from automated MUSCLE54 alignments of orthologous sequences within each cluster using HMMER55. The clusters described here are noted as ‘V4’ CyCOGs in the associated Data Records and on the ProPortal website48 (Data Citation 1).

Data Records

The complete dataset is available from the Prochlorococcus Portal website (Data Citation 1) and Dryad (Data Citation 2). The 27 Prochlorococcus genome sequences have also been deposited at DDBJ/EMBL/GenBank (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) under the accession numbers indicated in Table 2.

Datasets deposited at Dryad and ProPortal

Sequence, gene annotations, and COG definitions for Prochlorococcus genomes.

File 1—Tab-delimited file containing cluster assignments and annotation metadata for genes in the newly sequenced Prochlorococcus genomes described in this work, as well as previously published genomes. Columns are as follows:

Genome

The Prochlorococcus strain where the gene is found.

Gene ID

Unique ID for each Prochlorococcus gene, of the format ‘P<strain>_####’. Note that, due to the re-annotation of previously published genomes, these names (and the underlying gene boundaries) may not necessarily correspond to those in Genbank.

NCBI ID

For the new genome sequences presented here, the systematic NCBI locus_tag identifier for that gene. For previously published genomes, this column contains the corresponding Genbank locus ID (noted as an ‘Alternative locus ID’ for strains MED4, SS120 and MIT9313 in Genbank) from Kettler et al. (2007)4.

V1 CyCOG

Where applicable, the cyanobacterial cluster of orthologous groups of proteins (CyCOG) definition from Kettler et al. (2007)4.

V3 CyCOG

Where applicable, the CyCOG definition from Kelly et al. (2013)56.

V4 CyCOG

Number indicating the CyCOG to which this gene belongs, as defined in this work.

RAST annotation

Predicted functional annotation description, as supplied by the RAST annotation pipeline. Note that this text may differ slightly from the annotation in Genbank, due to changes imposed by NCBI annotation formatting guidelines.

GO annotation

Gene Ontology categorization for the gene, when available.

Argot2 annotation

Functional annotation prediction from the Argot2 pipeline, when available.

File 2 – Full RAST gene/protein sequence and annotation results. ZIP format file archive of individual tab-delimited files. Files are supplied for the new genome sequences presented here, as well as re-annotations of previously published genomes included in the CyCOG definitions. Columns are as follows:

contig_id

The name of the sequence contig on which the gene is found.

gene_id

The unique Gene ID code for that feature.

feature_id

Unique RAST-generated identifier for that feature.

type

peg: protein encoding gene; rna: RNA molecule.

location

Ordered location code for the position on the genome merging contig_id, start, and stop position.

start

Start location on contig, bp.

stop

Stop location on contig, bp.

strand

Orientation of gene on contig (+: on forward strand; −: on reverse).

function

The predicted function of the feature, if known.

aliases

Alternative names for the predicted function.

figfam

FigFAM membership for that feature.

evidence_codes

Code indicating the reason for the annotation. See http://www.nmpdr.org/FIG/wiki/view.cgi/FIG/EvidenceCode for more details.

nucleotide_sequence

The nucleotide sequence of the predicted gene.

aa_sequence

The protein (amino acid) sequence of the predicted gene.

File 3 – Set of nucleotide FASTA-formatted files containing the new Prochlorococcus genome assemblies described in this work.

File 4 – Set of nucleotide FASTA files containing all assembled contigs (>500 bp) from each culture (i.e., both Prochlorococcus and heterotrophs) sequenced in this work. Each file contains the set of contigs assembled from the raw sequencing data, before any filtering to separate Prochlorococcus from heterotroph contigs. These files are provided for reference, but due to the known heterotroph sequences in these files, they should be used with caution.

File 5 – Set of nucleotide FASTA files containing the predicted nucleotide sequence for all open reading frames (ORFs) in each genome. This file includes ORFs from both the new genomes presented here as well as the re-annotation of previously released Prochlorococcus genomes.

File 6 – Set of protein FASTA files containing the predicted amino acid translation for all ORFs in each genome. This file includes ORFs from both the new genomes presented here as well as the re-annotation of previously released Prochlorococcus genomes.

Technical Validation

Phylogenetic analysis of the ITS sequences obtained from these cultured isolate genomes (Figure 1) group these strains into the expected clades57 as previously determined from directed sequencing of the ITS sequences6. We were only able to obtain a single cyanobacterial ITS sequence from the assembled genome contigs, again consistent with these strains being unialgal. Prochlorococcus genome size and %GC content are typically quite similar for strains found within the same ITS-defined clade4, and both the draft and closed genomes are consistent with previously sequenced strains for these measures as well (Table 2).

The quality of the genome assemblies was assessed in multiple ways. Re-mapping of the original Illumina sequencing reads to the final assembled contigs showed that the reads were distributed evenly along the length of the assembly, ruling out some categories of major assembly errors (such as duplicated regions). Whole-genome alignments of contigs against closely related closed reference Prochlorococcus genomes indicated that the overall gene order of these contigs was broadly consistent with known sequences, indicating that the sequences do not contain obvious chimeras or other artifacts. We also estimated the completeness of the draft genomes by examining the core gene content of the strains, based on the set of genes shared by all closed Prochlorococcus genomes. We found that all of the draft genome assemblies contained >98% of the genes universally present in the 13 previously published closed Prochlorococcus genomes, indicating that these contigs represent most (or perhaps all) of the genome sequence.

The final closed sequences of the MIT0604 and MIT0801 genomes were verified in two additional ways. First, we compared the experimentally observed PCR product sizes from directed contig joining reactions to the distances predicted from the final assembled sequence to confirm the assembly. Second, we mapped the original (quality trimmed) Illumina sequencing reads against the final assembly. These alignments indicated that the final closed assembly was fully consistent with the original short-read sequence data. In addition, we confirmed that the per-base SNP frequency was not above the expected error frequency.

Additional information

How to cite this article: Biller, S. J. et al. Genomes of diverse isolates of the marine cyanobacterium Prochlorococcus. Sci. Data 1:140034 doi: 10.1038/sdata.2014.34 (2014).

Supplementary Material

sdata201434-isa1.zip (5KB, zip)

Acknowledgments

The authors are grateful to Allison Coe for careful maintenance of the MIT Prochlorococcus culture collection. We thank Luke Thompson, as well as the HOT and BATS teams, for assistance with field sampling. This work was supported in part by the Gordon and Betty Moore Foundation through Grant GBMF #495.01 and the National Science Foundation through grants OCE-1153588, OCE-0425602 and DBI-0424599, the NSF Center for Microbial Oceanography: Research and Education (C-MORE) to S.W.C. L.R.M. was supported by a NSF-ROA Supplement to NSF grant OCE-0806455 (to S.J.G.); L.R.M. and K.H.R.-J. were also supported by NSF OCE-0851288. G.R. was supported by NSF grant OCE-0723866.

Footnotes

The authors declare no competing financial interests.

Data Citations

  1. Biller S. J. 2014. Prochlorococcus Portal. http://proportal.mit.edu/
  2. Biller S. J. 2014. Dryad. http://dx.doi.org/10.5061/dryad.k282c
  3. Biller S. J. 2014. Genbank. JNAG00000000
  4. Biller S. J. 2014. Genbank. JNAH00000000
  5. Biller S. J. 2014. Genbank. CP007753
  6. Biller S. J. 2014. Genbank. JNAI00000000
  7. Biller S. J. 2014. Genbank. JNAJ00000000
  8. Biller S. J. 2014. Genbank. JNAK00000000
  9. Biller S. J. 2014. Genbank. JNAL00000000
  10. Biller S. J. 2014. Genbank. JNAM00000000
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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Citations

  1. Biller S. J. 2014. Prochlorococcus Portal. http://proportal.mit.edu/
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Supplementary Materials

sdata201434-isa1.zip (5KB, zip)

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