Methylobacterium fujisawaense Strain C14 Genomic Sequence
Julia Maresca, Iz Moxley, Jennifer Goff
Generated June 11, 2024

Methylobacterium fujisawaense Strain C14 Genomic Sequence

Accompanying Data for: "Genome sequence of Methylobacterium sp. strain C14, isolated from concrete"

Abstract

Methylobacterium fujisawaense strain C14 was isolated from a weathered concrete cylinder. Using PacBio sequencing, a high-quality genome secquence was generated for strain C14 to facilitate the development genetic tools for engineering of microorganisms inhabiting concrete.

Introduction

The publication by E. Anders Kiledal, Iz Moxley, Olga Shevchenko, Jennifer L. Goff, Julia A. Maresca can be found here: [URL].

The C14 strain was isolated from a concrete cylinder that had weathered outside for 16 months as part of a long-term study on the microbiome of weathered concrete (Ref Kiledal 2021). The cylinder was sliced into ~2-cm slices using a saw with an alcohol-sterilized blade, then slices were broken with a hammer. Small (Less than 2cm^3) concrete pieces were placed in 30 mL phosphate-buffered saline (ThermoFisher, 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4) with 0.5% TWEEN-20, sonicated in a bath sonicator for 5 minutes, shaken in an incubator at 25°C and ~180 rpm for 2 hours, then plated. The medium was 10% standard BG-11 (Allen & Stanier 1968) solidified with gellan gum and 8 mM CaCl2, amended with 2.5 g L-1 each peptone, yeast extract, and glucose.

Table of Contents

  1. Background and Experimental Methods
  2. Import and annotation
  3. QC, Assembly, and Annotation
  4. Taxonomic Classification
  5. References
Narrative created by: Iz Moxley

Background and Experimental Methods

Strain C14 was isolated from a concrete cylinder that had weathered outside for 15 months as part of a long-term study on the microbiome of weathered concrete (1). Because other Methylobacterium spp. are genetically tractable (2), we sequenced its genome as part of an effort to develop genetic tools for bacterial isolates from concrete. Prior to genome sequencing, strain C14 was revived from the freezer stock on LB medium (1.5% agar) and incubated at room temperature until colonies appeared. A single colony was inoculated into 10 mL LB and incubated with shaking at room temperature until stationary phase. DNA was extracted using a phenol-chloroform extraction protocol optimized for Gram-positive bacteria (4). A single-molecule real-time (SMRT) library was barcoded and prepared using the PacBio SMRTbell Express template preparation kit version 2.0. DNA fragments larger than 6 kb were size selected using BluePippin (Sage Science). The average library fragment size was 12 kb, as measured by a fragment analyzer (Advanced Analytical Technologies, Inc.). Sequencing was completed on a PacBio Sequel IIe single-molecule sequencer in one 1M version 3 LR SMRT Cell with a 30-h movie. Samples were demultiplexed using PacBio SMRT Link version 11. The PacBio sequencing yielded 55,332 raw reads with an N50 of 12,235 bp.

Import

The Methylobacterium genome was imported using the globus application and unpacked using Unpack a Compressed File in Staging Area.

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": "demultiplex.C14.hifi_reads.fastq",
            "fastq_rev_staging_file_name": None,
            "name": "C14_reads"
        }],
        "shared_params": {
            "sequencing_tech": "PacBio CCS",
            "single_genome": 1,
            "read_orientation_outward": 0,
            "insert_size_std_dev": None,
            "insert_size_mean": None
        }
    }],
    cell_id="6744313f-f7cc-48e5-8d22-c9adeb926dde",
    run_id="f585d956-99dd-469f-bd9d-e48a5cfb6881"
)
flye_C14jm.contigs
v1 - KBaseGenomeAnnotations.Assembly-5.1
The viewer for the data in this Cell is available at the original Narrative here: https://narrative.kbase.us/narrative/169030
Filter Reads with Filtlong - v0.2.1
Filter long reads using Filtlong.
This app completed without errors in 2m 16s.
Objects
Created Object Name Type Description
C14_filtlong.reads SingleEndLibrary Filtered reads
Summary
Running run_kb_filtlong with params: { "workspace_name": "jmaresca:narrative_1706555514993", "workspace_id": 169030, "input_reads_library": "169030/2/1", "input_short_paired_library": null, "output_reads_name": "C14_filtlong.reads", "min_read_length": 1000, "keep_percent": 90, "target_bases": 500000000 } Getting long reads (from reads library object). command: filtlong --min_length 1000 --keep_percent 90 --target_bases 500000000 /kb/module/work/tmp/23747df1-86b7-4a64-ac4a-7c7f4e517ddd.single.fastq > /kb/module/work/tmp/filtlong_output_9e50aab2-5167-427e-ada1-2424654ccdfc.fastq Scoring long reads 37 reads (484908 bp) 76 reads (972107 bp) 115 reads (1463849 bp) 154 reads (1955845 bp) 194 reads (2445352 bp) 231 reads (2932256 bp) 270 reads (3417855 bp) 309 reads (3909314 bp) 349 reads (4395021 bp) 390 reads (4882733 bp) 430 reads (5371147 bp) 470 reads (5863941 bp) 508 reads (6348270 bp) 548 reads (6837080 bp) 590 reads (7320761 bp) 629 reads (7806043 bp) 670 reads (8305255 bp) 707 reads 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(651185850 bp) 52898 reads (651682007 bp) 52938 reads (652176381 bp) 52977 reads (652662823 bp) 53017 reads (653148117 bp) 53056 reads (653636168 bp) 53096 reads (654128065 bp) 53136 reads (654616548 bp) 53175 reads (655106572 bp) 53217 reads (655598266 bp) 53258 reads (656087728 bp) 53298 reads (656578823 bp) 53336 reads (657069826 bp) 53377 reads (657556072 bp) 53416 reads (658045116 bp) 53457 reads (658535514 bp) 53496 reads (659020131 bp) 53535 reads (659507353 bp) 53575 reads (659999990 bp) 53615 reads (660486877 bp) 53656 reads (660974554 bp) 53694 reads (661461059 bp) 53734 reads (661945001 bp) 53777 reads (662441102 bp) 53816 reads (662925277 bp) 53858 reads (663413418 bp) 53900 reads (663907387 bp) 53939 reads (664411581 bp) 53980 reads (664895979 bp) 54020 reads (665386338 bp) 54059 reads (665876211 bp) 54099 reads (666362534 bp) 54141 reads (666848663 bp) 54180 reads (667341010 bp) 54221 reads (667834268 bp) 54260 reads (668325793 bp) 54299 reads (668816516 bp) 54338 reads (669301341 bp) 54376 reads (669786129 bp) 54418 reads (670275164 bp) 54457 reads (670763491 bp) 54498 reads (671264812 bp) 54536 reads (671751456 bp) 54573 reads (672240800 bp) 54610 reads (672730546 bp) 54652 reads (673218334 bp) 54693 reads (673707731 bp) 54733 reads (674198079 bp) 54770 reads (674681981 bp) 54811 reads (675172716 bp) 54852 reads (675663074 bp) 54894 reads (676158954 bp) 54934 reads (676646239 bp) 54971 reads (677130122 bp) 55010 reads (677624719 bp) 55049 reads (678115096 bp) 55091 reads (678599177 bp) 55132 reads (679084794 bp) 55170 reads (679572179 bp) 55211 reads (680061214 bp) 55253 reads (680550908 bp) 55295 reads (681043288 bp) 55332 reads (681486930 bp) Filtering long reads target: 500000000 bp keeping 500000056 bp Outputting passed long reads Uploading filtered reads: C14_filtlong.reads Generating and saving report. Filtlong results saved.
Assemble Long Reads with Flye - v2.9.2
Assemble long reads using the Flye assembler.
This app completed without errors in 39m 36s.
Objects
Created Object Name Type Description
C14_flye.contigs Assembly Assembled contigs
Report
Summary
Flye results saved to: jmaresca:narrative_1706555514993//kb/module/work/tmp/flye_2b6c12e7-cebe-4a6d-a52c-0872457a5ecb Assembly saved to: jmaresca:narrative_1706555514993/C14_flye.contigs Assembled into 9 contigs. Avg Length: 788296.4444444445 bp. Contig Length Distribution (# of contigs -- min to max basepairs): 8 -- 33746.0 to 696044.5 bp 0 -- 696044.5 to 1358343.0 bp 0 -- 1358343.0 to 2020641.5 bp 0 -- 2020641.5 to 2682940.0 bp 0 -- 2682940.0 to 3345238.5 bp 0 -- 3345238.5 to 4007537.0 bp 0 -- 4007537.0 to 4669835.5 bp 0 -- 4669835.5 to 5332134.0 bp 0 -- 5332134.0 to 5994432.5 bp 1 -- 5994432.5 to 6656731.0 bp
Links
Files
These are only available in the live Narrative: https://narrative.kbase.us/narrative/169030
  • flye_output.zip - Output file(s) generated by Flye
C14_flye.contigs
v1 - KBaseGenomeAnnotations.Assembly-5.1
The viewer for the data in this Cell is available at the original Narrative here: https://narrative.kbase.us/narrative/169030

QC, Assembly, and Annotation

Assembly and Annotation

Assembly and annotation of the strain C14 genome were performed using KBase (5). Filtlong v0.2.1 (6) (kb_flye v0.2.2) was used to filter reads that were <1000 bp. After filtering, 35,425 reads remained. These reads were assembled and polished with Flye v2.9.2 (7)(kb_flye v0.2.2). The Flye log was checked for circularity. Mobile genetic elements were predicted using geNomad v1.7.5 (9) implemented in NMDC-EDGE (10). The strain C14 genome was annotated using RASTtk v1.073 (11)(RAST_SDK v1.9.5), and the resulting annotations are available in the accompanying KBase narrative. There are 9 contigs, 1 chromosome, and one suspected phage. The GC content is 69.5%. The strain C14 genome was deposited in GenBank and reannotated using the Prokaryotic Genome Annotation Pipeline (PGAP) v## (12). Default parameters were used except where otherwise noted.

A summary of the geNomad results is shown below:

Viral Predictions
contig length (bp) coordinates virus score
contig_1 (provirus) 65,472 70-65,541 0.979
contig_1 (provirus) 55,857 5,715,213-5,771,069 0.953
contig_1 (provirus) 56,316 6,161,344-6,217,659 0.952
contig_3 33,746 NA 0.907
Plasmid Predictions
contig length (bp) plasmid score
contig_9 54,537 0.988
contig_8 85,428 0.987
contig_7 76,184 0.986
contig_2 62,136 0.982
contig_5 51,201 0.979
contig_6 39,253 0.976
contig_4 35,452 0.965

Quality Control

Genome completeness and contamination were estimated at 100.0% and 0.0%, using CheckM v1.0.18 (8) (kb_Msuite v1.4.2) and using default parameters.

Assess Genome Quality with CheckM - v1.0.18
Runs the CheckM lineage workflow to assess the genome quality of isolates, single cells, or genome bins from metagenome assemblies through comparison to an existing database of genomes.
This app completed without errors in 5m 39s.
Report
Links
Files
These are only available in the live Narrative: https://narrative.kbase.us/narrative/169030
  • CheckM_summary_table.tsv.zip - TSV Summary Table from CheckM
  • full_output.zip - Full output of CheckM
  • plots.zip - Output plots from CheckM
Annotate Genome/Assembly with RASTtk - v1.073
Annotate or re-annotate genome/assembly using RASTtk (Rapid Annotations using Subsystems Technology toolkit).
This app completed without errors in 10m 5s.
Objects
Created Object Name Type Description
C14_annotation_jm Genome RAST re-annotated genome
Report
Summary
The RAST algorithm was applied to annotating a genome sequence comprised of 9 contigs containing 7094668 nucleotides. No initial gene calls were provided. Standard features were called using: glimmer3; prodigal. A scan was conducted for the following additional feature types: rRNA; tRNA; selenoproteins; pyrrolysoproteins; repeat regions; crispr. The genome features were functionally annotated using the following algorithm(s): Kmers V2; Kmers V1; protein similarity. In addition to the remaining original 0 coding features and 0 non-coding features, 7624 new features were called, of which 246 are non-coding. Output genome has the following feature types: Coding gene 7378 Non-coding repeat 179 Non-coding rna 67 The number of distinct functions can exceed the number of genes because some genes have multiple functions.
Links

Taxonomic Identification

The Strain C14 is most closely related to Methylobacterium fujisawaense strain C14 (98.19% ANI calculated by GTDB-TK v. 1.7.0), in the class Alphaproteobacteria.

Classify Microbes with GTDB-Tk - v1.7.0
Obtain objective taxonomic assignments for bacterial and archaeal genomes based on the Genome Taxonomy Database (GTDB) ver R06-RS202
This app completed without errors in 36m 2s.
Report
Links
Insert Genome Into SpeciesTree - v2.2.0
Add one or more Genomes to a KBase SpeciesTree.
This app completed without errors in 4m 32s.
Report
Links
Files
These are only available in the live Narrative: https://narrative.kbase.us/narrative/169030
  • Methylorubum-C14Tree.newick
  • Methylorubum-C14Tree-labels.newick
  • Methylorubum-C14Tree.png
  • Methylorubum-C14Tree.pdf
Methylorubum-C14Tree
v1 - KBaseTrees.Tree-1.0
The viewer for the data in this Cell is available at the original Narrative here: https://narrative.kbase.us/narrative/169030
C14Genomeset
v1 - KBaseSearch.GenomeSet-2.1
The viewer for the data in this Cell is available at the original Narrative here: https://narrative.kbase.us/narrative/169030

References

  1. Kiledal EA, Keffer JL, Maresca JA. 2021. Bacterial Communities in Concrete Reflect Its Composite Nature and Change with Weathering. mSystems 6:10.1128/msystems.01153-20.

  2. Marx CJ, Lidstrom ME. 2001. Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteriaThe GenBank accession numbers for the sequences reported in this paper are AF327711, AF327712, AF327713, AF327714, AF327715, AF327716, AF327717, AF327718, AF327719 and AF327720. Microbiology 147:2065-2075.

  3. Allen MM, Stanier RY. 1968. Growth and Division of Some Unicellular Blue-green Algae. Microbiology 51:199-202.

  4. Kiledal E, Maresca JA. 2021. Chromosomal DNA extraction from Gram-positive bacteria. protocolsio.

  5. Arkin AP, Cottingham RW, Henry CS, Harris NL, Stevens RL, Maslov S, Dehal P, Ware D, Perez F, Canon S, Sneddon MW, Henderson ML, Riehl WJ, Murphy-Olson D, Chan SY, Kamimura RT, Kumari S, Drake MM, Brettin TS, Glass EM, Chivian D, Gunter D, Weston DJ, Allen BH, Baumohl J, Best AA, Bowen B, Brenner SE, Bun CC, Chandonia J-M, Chia J-M, Colasanti R, Conrad N, Davis JJ, Davison BH, DeJongh M, Devoid S, Dietrich E, Dubchak I, Edirisinghe JN, Fang G, Faria JP, Frybarger PM, Gerlach W, Gerstein M, Greiner A, Gurtowski J, Haun HL, He F, Jain R, et al. 2018. KBase: The United States Department of Energy Systems Biology Knowledgebase. Nat Biotechnol 36:566-569.

  6. Wick R. Filtlong https://github. com/rrwick. Filtlong.

  7. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. 2019. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 37:540-546.

  8. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. 2015. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 25:1043-1055.

  9. Camargo AP, Roux S, Schulz F, Babinski M, Xu Y, Hu B, Chain PS, Nayfach S, Kyrpides NC. 2023. Identification of mobile genetic elements with geNomad. Nat Biotechnol:1-10.

  10. Eloe-Fadrosh EA, Ahmed F, Babinski M, Baumes J, Borkum M, Bramer L, Canon S, Christianson DS, Corilo YE, Davenport KW. 2022. The National Microbiome Data Collaborative Data Portal: an integrated multi-omics microbiome data resource. Nucleic Acids Res 50:D828-D836.

  11. Brettin T, Davis JJ, Disz T, Edwards RA, Gerdes S, Olsen GJ, Olson R, Overbeek R, Parrello B, Pusch GD, Shukla M, Thomason JA, Stevens R, Vonstein V, Wattam AR, Xia F. 2015. RASTtk: A modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep 5:8365.

  12. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res 44:6614-24.

  13. Chaumeil P-A, Mussig AJ, Hugenholtz P, Parks DH. 2020. GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database. Bioinformatics 36:1925-1927.

  14. Green P, Bousfield I, Hood D. 1988. Three new Methylobacterium species: M. rhodesianum sp. nov., M. zatmanii sp. nov., and M. fujisawaense sp. nov. Int J Syst Evol Microbiol 38:124-127.

Released Apps

  1. Assess Genome Quality with CheckM - v1.0.18
    • Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015;25: 1043 1055. doi:10.1101/gr.186072.114
    • CheckM source:
    • Additional info:
  2. Insert Genome Into SpeciesTree - v2.2.0
    • Price MN, Dehal PS, Arkin AP. FastTree 2 Approximately Maximum-Likelihood Trees for Large Alignments. PLoS One. 2010;5. doi:10.1371/journal.pone.0009490

Apps in Beta

  1. Annotate Genome/Assembly with RASTtk - v1.073
    • [1] Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST Server: Rapid Annotations using Subsystems Technology. BMC Genomics. 2008;9: 75. doi:10.1186/1471-2164-9-75
    • [2] Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, et al. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res. 2014;42: D206 D214. doi:10.1093/nar/gkt1226
    • [3] Brettin T, Davis JJ, Disz T, Edwards RA, Gerdes S, Olsen GJ, et al. RASTtk: A modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep. 2015;5. doi:10.1038/srep08365
    • [4] Kent WJ. BLAT The BLAST-Like Alignment Tool. Genome Res. 2002;12: 656 664. doi:10.1101/gr.229202
    • [5] Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25: 3389-3402. doi:10.1093/nar/25.17.3389
    • [6] Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997;25: 955 964.
    • [7] Cobucci-Ponzano B, Rossi M, Moracci M. Translational recoding in archaea. Extremophiles. 2012;16: 793 803. doi:10.1007/s00792-012-0482-8
    • [8] Meyer F, Overbeek R, Rodriguez A. FIGfams: yet another set of protein families. Nucleic Acids Res. 2009;37 6643-54. doi:10.1093/nar/gkp698.
    • [9] van Belkum A, Sluijuter M, de Groot R, Verbrugh H, Hermans PW. Novel BOX repeat PCR assay for high-resolution typing of Streptococcus pneumoniae strains. J Clin Microbiol. 1996;34: 1176 1179.
    • [10] Croucher NJ, Vernikos GS, Parkhill J, Bentley SD. Identification, variation and transcription of pneumococcal repeat sequences. BMC Genomics. 2011;12: 120. doi:10.1186/1471-2164-12-120
    • [11] Hyatt D, Chen G-L, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010;11: 119. doi:10.1186/1471-2105-11-119
    • [12] Delcher AL, Bratke KA, Powers EC, Salzberg SL. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics. 2007;23: 673 679. doi:10.1093/bioinformatics/btm009
    • [13] Akhter S, Aziz RK, Edwards RA. PhiSpy: a novel algorithm for finding prophages in bacterial genomes that combines similarity- and composition-based strategies. Nucleic Acids Res. 2012;40: e126. doi:10.1093/nar/gks406
  2. Assemble Long Reads with Flye - v2.9.4
    • [1] Mikhail Kolmogorov, Jeffrey Yuan, Yu Lin and Pavel Pevzner, "Assembly of Long Error-Prone Reads Using Repeat Graphs", Nature Biotechnology, 2019 doi:10.1038/s41587-019-0072-8
  3. Classify Microbes with GTDB-Tk - v1.7.0
    • Pierre-Alain Chaumeil, Aaron J Mussig, Philip Hugenholtz, Donovan H Parks, GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database, Bioinformatics, Volume 36, Issue 6, 15 March 2020, Pages 1925 1927. DOI: https://doi.org/10.1093/bioinformatics/btz848
    • Parks, D., Chuvochina, M., Waite, D. et al. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nat Biotechnol 36, 996 1004 (2018). DOI: https://doi.org/10.1038/nbt.4229
    • Parks DH, Chuvochina M, Chaumeil PA, Rinke C, Mussig AJ, Hugenholtz P. A complete domain-to-species taxonomy for Bacteria and Archaea. Nat Biotechnol. 2020;10.1038/s41587-020-0501-8. DOI:10.1038/s41587-020-0501-8
    • Rinke C, Chuvochina M, Mussig AJ, Chaumeil PA, Dav n AA, Waite DW, Whitman WB, Parks DH, and Hugenholtz P. A standardized archaeal taxonomy for the Genome Taxonomy Database. Nat Microbiol. 2021 Jul;6(7):946-959. DOI:10.1038/s41564-021-00918-8
    • Matsen FA, Kodner RB, Armbrust EV. pplacer: linear time maximum-likelihood and Bayesian phylogenetic placement of sequences onto a fixed reference tree. BMC Bioinformatics. 2010;11:538. Published 2010 Oct 30. doi:10.1186/1471-2105-11-538
    • Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun. 2018;9(1):5114. Published 2018 Nov 30. DOI:10.1038/s41467-018-07641-9
    • Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010;11:119. Published 2010 Mar 8. DOI:10.1186/1471-2105-11-119
    • Price MN, Dehal PS, Arkin AP. FastTree 2--approximately maximum-likelihood trees for large alignments. PLoS One. 2010;5(3):e9490. Published 2010 Mar 10. DOI:10.1371/journal.pone.0009490 link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835736/
    • Eddy SR. Accelerated Profile HMM Searches. PLoS Comput Biol. 2011;7(10):e1002195. DOI:10.1371/journal.pcbi.1002195
  4. Filter Reads with Filtlong - v0.2.1
    • [1] Wick RR, 2017. Filtlong.