Generated March 12, 2024
This is the Narrative for Porphyromonadaceae sp. W3.11. A complementary Narrative for Lachnospiraceae sp. C1.1 is available here.
This is the Narrative for Lachnospiraceae sp. C1.1. A complementary Narrative for Porphyromonadaceae sp. W3.11 is available here.
This Narrative and its complementary Narrative contain assembly and annotation of two bacterial isolates that were isolated by our laboratory from the rumen of a Holstein heifer. All procedures with animals have been approved by University of California Davis’s Institutional Animal Care and Use Committee. Rumen contents were collected through a rumen fistula and strained through two layers of cheesecloth into a bottle. The bottle was sealed to exclude air and maintained at 39°C. Contents were brought to the laboratory and bubbled under O2-free CO2 within 15 min. At the laboratory, serial dilutions were made with anaerobic dilution solution for Lachnospiraceae sp. C1.1 and propionibacterium diluent for Porphyromonadaceae sp. W3.11 (table S2). Aliquots (0.1 ml) of each dilution were injected into anaerobic bottle plates (1) containing 9 ml of LH medium (table S2). After incubation at 37°C for 7 days, isolated colonies were picked. Lachnospiraceae sp. C1.1 was picked from a bottle inoculated with a 104 dilution of rumen contents, and Porphyromonadaceae sp. W3.11 was picked from a bottle inoculated with a 103 dilution. After initial isolation, these organisms were purified by growing on anaerobic roll tubes (2) and picking isolated colonies.
We performed de novo sequencing of Lachnospiraceae sp. C1.1 and Porphyromonadaceae sp. W3.11. Aliquots of liquid culture (9 and 1.5 ml, respectively) were collected by syringe and centrifuged (21,000g for 10 min at 4°C). Cell pellets were submitted to Molecular Research LP for DNA extraction, library preparation, and sequencing. After resuspending pellets in 180 µl of ATL buffer (Qiagen), DNA was extracted using the MagAttract HMW DNA Kit (Qiagen). DNA was eluted in 100 µl of AE buffer (Qiagen) and then cleaned using the DNEasy PowerClean Pro Cleanup Kit (Qiagen). DNA was then sheared using the Covaris g-TUBE (Covaris). Sequencing libraries were prepared using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences) and 1500 ng of the sheared and purified DNA. The SMRTbell libraries were size-selected (>6 Kb) using a BluePippin instrument (Sage Science) and 0.75% agarose gel. Libraries were then sequenced using the PacBio Sequel II (Pacific Biosciences) platform and a 30-hour movie time.
In these Narratives, we filtered low-quality reads using Trimmomatic (v0.36), assembled filtered reads with SPAdes (v3.15.3), and then checked completeness and contamination of the assembled genomes with CheckM (v1.0.18). Statistics for sequencing and assembly are in table S3.
Using the assembled contigs (genomes), we called genes and annotated them. Protein-coding genes were called using Prodigal (v2.6.3) (3) locally or using KBase via RASTtk (v1.073), with identical results. Genes were annotated with KO IDs using KAAS (4). They were further annotated with pfam and TIGRFAM IDs using KBase and the Annotate Domains in a Genome app. We classified putative genes for hydrogenases using HydDB. Genes for 16S ribosomal RNA (rRNA) were called using RASTtk (v1.073) in KBase.
The contigs (genomes) were analyzed to determine whether they belonged to new species. Taxonomy was assigned using GTDB-Tk (v1.7.0) in KBase. The identity of 16S rRNA genes to other organisms was found using EzBioCloud (5). Values of digital DNA-DNA hybridization (dDDH) were found with Type (Strain) Genome Server (6). These analyses suggest that Lachnospiraceae sp. C1.1 and Porphyromonadaceae sp. W3.11 represent novel species or genera. GTDB-Tk assigned Lachnospiracae sp. C1.1 to family Lachnospiraceae and genus NK4A144, which contains no type strains. It assigned Porphyromonadaceae sp. W3.11 to Porphyromonadaceae and genus Porphyromonas_A. Values of 16S rRNA identity and dDDH with respect to type strains were low (table S4). Although more phenotypic data are needed, available evidence supports assignment of genomes to new species or genera.
Hackmann TJ, Zhang B. The phenotype and genotype of fermentative prokaryotes. Sci Adv. 2023 Sep 29;9(39):eadg8687. doi: 10.1126/sciadv.adg8687. Epub 2023 Sep 27. PMID: 37756392; PMCID: PMC10530074.
Supplementary tables referenced above can be downloaded here.
from biokbase.narrative.jobs.appmanager import AppManager
AppManager().run_app_batch(
[{
"app_id": "kb_uploadmethods/import_fastq_noninterleaved_as_reads_from_staging",
"tag": "release",
"version": "5b9346463df88a422ff5d4f4cba421679f63c73f",
"params": [{
"fastq_fwd_staging_file_name": "W3.11.fastq",
"fastq_rev_staging_file_name": None,
"name": "W3.11"
}],
"shared_params": {
"sequencing_tech": "PacBio CCS",
"single_genome": 1,
"read_orientation_outward": 0,
"insert_size_std_dev": None,
"insert_size_mean": None
}
}],
cell_id="e6ecbf83-3880-4680-82ea-fa2a57623010",
run_id="1c2243f0-35d7-4f9e-bbb8-de604c6cd364"
)
| Created Object Name | Type | Description |
|---|---|---|
| W3.11_trimmed | SingleEndLibrary | Trimmed Reads |
| W3.11_trimmed |
| Created Object Name | Type | Description |
|---|---|---|
| SPAdes.Assembly_trimmed | Assembly | Assembled contigs |
| Created Object Name | Type | Description |
|---|---|---|
| W3.11_genes | Genome | RAST annotation |
The RAST algorithm was applied to annotating a genome sequence comprised of 9 contigs containing 2628695 nucleotides. No initial gene calls were provided. Standard gene features were called using: prodigal. In addition to the remaining original 0 coding features and 0 non-coding features, 2256 new features were called, of which 0 are non-coding. Output genome has the following feature types: Coding gene 2256 Overall, the genes have 0 distinct functions. The genes include 0 genes with a SEED annotation ontology across 0 distinct SEED functions. The number of distinct functions can exceed the number of genes because some genes have multiple functions.
| Created Object Name | Type | Description |
|---|---|---|
| W3.11_annotations | DomainAnnotation | Domain Annotations |
| Created Object Name | Type | Description |
|---|---|---|
| W3.11_rRNA | Genome | RAST annotation |
The RAST algorithm was applied to annotating a genome sequence comprised of 9 contigs containing 2628695 nucleotides. No initial gene calls were provided. A scan was conducted for the following additional feature types: rRNA. In addition to the remaining original 0 coding features and 0 non-coding features, 6 new features were called, of which 6 are non-coding. Output genome has the following feature types: Non-coding rna 6 Overall, the genes have 0 distinct functions. The genes include 0 genes with a SEED annotation ontology across 0 distinct SEED functions. The number of distinct functions can exceed the number of genes because some genes have multiple functions.
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R. Hungate, Chapter IV A roll tube method for cultivation of strict anaerobes. Methods Microbiol. 3, 117–132 (1969). doi: 10.1016/S0580-9517(08)70503-8
D. Hyatt, G. L. Chen, P. F. LoCascio, M. L. Land, F. W. Larimer, L. J. Hauser, Prodigal: Prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11, 119 (2010). doi:10.1186/1471-2105-11-119
Y. Moriya, M. Itoh, S. Okuda, A. C. Yoshizawa, M. Kanehisa, KAAS: An automatic genome annotation and pathway reconstruction server. Nucleic Acids Res. 35, W182–W185 (2007). doi:10.1093/nar/gkm321
S. H. Yoon, S. M. Ha, S. Kwon, J. Lim, Y. Kim, H. Seo, J. Chun, Introducing EzBioCloud: A taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int. J. Syst. Evol. Microbiol. 67, 1613–1617 (2017).doi:10.1099/ijsem.0.001755
J. P. Meier-Kolthoff, J. S. Carbasse, R. L. Peinado-Olarte, M. Goker, TYGS and LPSN: A database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes. Nucleic Acids Res. 50, D801–D807 (2022). doi: 10.1093/nar/gkab902