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Complete genome sequence of Agrobacterium fabrum ARqua1

October 2023
Microbiology Resource Announcements 12(11)

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We report the complete genome of Agrobacterium fabrum ARqua1 generated from Oxford Nanopore and Illumina sequencing. The genome of ARqua1 has a total length of 5,714,310 bp, comprising a circular chromosome, a linear chromosome, and two plasmids. In total, 5,446 genes were predicted, of which 5,288 were annotated.

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| Biotechnology | Announcement

Complete genome sequence of Agrobacterium fabrum ARqua1

Bo Lan,1 Qian Zhang,2 Kangquan Yin1

AUTHOR AFFILIATIONS See aliation list on p. 2.

ABSTRACT We report the complete genome of Agrobacterium fabrum ARqua1

generated from Oxford Nanopore and Illumina sequencing. The genome of ARqua1 has

a total length of 5,714,310 bp, comprising a circular chromosome, a linear chromosome,

and two plasmids. In total, 5,446 genes were predicted, of which 5,288 were annotated.

KEYWORDS genomes, Agrobacterium, genome editing

Agrobacterium fabrum is a well-known natural agent that induces hairy root diseases

in plants and has been widely used in transgenic technology (1–3). Among these, A.

fabrum ARqua1 has been used for genetic transformation in more than 60 plant species

(4). The ARqua1 is derived from strain R1000, which has the C58 chromosomal back

ground from A. tumefaciens A136 (5). Recently, a draft shotgun assembly of A. fabrum

ARqua1 consisting of 19 contigs was reported (6). This report describes the complete

genome assembly of A. fabrum ARqua1, which will facilitate genetic engineering of the

“engineer” and generation of improved Agrobacterium strains for plant transformation

and genome editing (7).

ARqua1 strain was obtained from Shanghai Weidi Biotechnology Co. Ltd. The strain

was streaked onto Luria broth (LB) plates, and a single colony was selected for

amplication with streptomycin (100 mg/L). The same genomic DNA extracted using

the cetyltrimethylammonium bromide (CTAB) method (8) was used for both Oxford

Nanopore and Illumina sequencing. For Oxford Nanopore sequencing, DNA was sheared

using g-TUBE, and large DNA fragments (>20 Kb) were selected using BluePippin (Sage

Science, Beverly, MA). Nanopore libraries were prepared using the SQK-LSK109 Genomic

DNA Ligation Kit (Oxford Nanopore Technologies, UK) and sequenced on an FLO-PRO002

R9.4.1 ow cell. The basecalling and adapter trimming were performed using Guppy

version 3.2.6. Read QC was performed using fastp v0.23.1 (9). Reads with quality scores

less than 6.0 or shorter than 2,000 bp were ltered. Nanopore sequencing yielded

101,392 clean reads and an N50 of 26,601 bp. Illumina libraries were prepared using

the TrueLib DNA Library Rapid Prep Kit and sequenced on an Illumina NovaSeq 6000

(Illumina, San Diego, CA, USA). Illumina sequencing yielded 12,333,606 paired-end reads

(mean read length, 147 bp), which were trimmed using fastp version 0.23.1 (9), resulting

in a total of 988,367,215 bp (~173-fold coverage). Nanopore sequencing data were used

for de novo genome assembly using the Canu version 1.5 (10), and the post-assembly

correction was conducted using Racon version 3.4.3 (11). Illumina sequencing data were

used for further correction using Pilon version 1.22 (12). Circularization of chromosomal

and plasmid contigs was conducted using Circlator version 1.5.5 (13) with the parameter

“minimus2 --no_pre_merge.” Genome was annotated using Prodigal version 2.6.3 (14),

RepeatMasker version 4.0.5 (15), Infernal version 1.1.3 (16), and tRNAscan-SE version

2.0 (17), respectively. Functional annotation of genes was conducted using eggNOG

version 4.0, Pfam version 32.0, Swissprot access date 2019-07-31, and TrEMBL access date

2019-07-13 databases. Collinearity analysis was performed using the One Step MCScanX

plugin in TBtools version 1.120 (18). Default parameters were used for all of the above

software unless otherwise specied.

November 2023 Volume 12 Issue 11 10.1128/MRA.00554-23 1

Editor J. Cameron Thrash, University of Southern

California, Los Angeles, California, USA

Address correspondence to Kangquan Yin,

yinkq@bjfu.edu.cn.

The authors declare no conict of interest.

See the funding table on p. 3.

Received 29 June 2023

Accepted 22 August 2023

Published 9 October 2023

article distributed under the terms of the Creative

Commons Attribution 4.0 International license.

The assembly resulted in four contigs: a circular chromosome, a linear chromosome,

and two plasmids. The assembled genome had a GC content of 59.03%. Collinearity

analysis showed that contig4 of the current ARqua1 genome was plasmid pRiA4b and

conrmed that the chromosomal background of the ARqua1 strain was A. fabrum C58

(Fig. 1).

ACKNOWLEDGMENTS

This work was supported by grants from the Fundamental Research Funds for the Central

Universities (2021ZY80) and Science and Technology Innovation of Inner Mongolia

Autonomous Region (2022JBGS0020).

AUTHOR AFFILIATIONS

1School of Grassland Science, Beijing Forestry University, Beijing, Beijing, China

2Chinese Academy of Agricultural Sciences, Lanzhou Institute of Husbandry and

Pharmaceutical Sciences, Lanzhou, China

AUTHOR ORCIDs

Kangquan Yin http://orcid.org/0000-0002-4627-6585

A. fabrum ARqua1

(this study)

4321

GCA_000092025.1

Contig

Contig A. fabrum ARqua1

(this study)

4321

1151613271512391761410814 18

GCA_012971785.1

GCA_018138105.1

Chr2Chr1 pRiA4pArA4

pAtC58 pTiC58

Chr2Chr1

Contig

FIG 1 Genome synteny maps. (a) Synteny links between current A. fabrum ARqua1 genome and A. rhizogenes A4 genome (GCA_018138105.1, https://

www.ncbi.nlm.nih.gov/datasets/genome/GCF_018138105.1/), and synteny links between current A. fabrum ARqua1 genome and A. fabrum C58 genome

(GCA_000092025.1, https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000092025.1/). The red lines indicate the synteny links between the plasmid pRiA4 of

A. rhizogenes A4 and contig 4 of the current A. fabrum ARqua1 genome. (b) Synteny links between the current A. fabrum ARqua1 genome and the rst draft

genome of A. fabrum ARqua1 (GCA_012971785.1, https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_012971785.1/). The blue lines indicate the syntenic links

between contig 11 of the draft genome of A. fabrum ARqua1 and contig 4 of the current A. fabrum ARqua1 genome.

Announcement Microbiology Resource Announcements

November 2023 Volume 12 Issue 11 10.1128/MRA.00554-23 2

FUNDING

Funder Grant(s) Author(s)

MOE | Fundamental Research Funds for the Central

Universities (Fundamental Research Fund for the Central

Universities)

2021ZY80 Kangquan Yin

Science and Technology Innovation of Inner Mongolia

Autonomous Region

2022JBGS0020 Kangquan Yin

AUTHOR CONTRIBUTIONS

Bo Lan, Conceptualization, Data curation, Formal analysis, Funding acquisition,

Investigation, Methodology, Project administration, Resources, Supervision, Writing –

original draft, Writing – review and editing | Qian Zhang, Data curation, Resources,

Writing – original draft | Kangquan Yin, Conceptualization, Data curation, Formal analysis,

Funding acquisition, Investigation, Methodology, Project administration, Resources,

Supervision, Writing – original draft, Writing – review and editing

DATA AVAILABILITY

The Bioproject number is PRJNA976066. The SRA accession numbers are SRR24759617

(Illumina) and SRR24759618 (ONT).

REFERENCES

1. Chilton MD, Tepfer DA, Petit A, David C, Casse-Delbart F, Tempé J. 1982.

Agrobacterium rhizogenes inserts T-DNA into the genomes of the host

plant root cells. Nature 295:432–434. https://doi.org/10.1038/295432a0

2. OzyigitII, Dogan I, Artam TE. 2013. Agrobacterium rhizogenes mediated

transformation and its biotechnological applications in crops, p 1–48. In

Hakeem RK, P Ahmad, M Ozturk (ed), Crop improvement: new

approaches and modern techniques. Springer, Boston. https://doi.org/

10.1007/978-1-4614-7028-1

3. Yin K, Gao C, Qiu JL. 2017. Progress and prospects in plant genome

editing. Nat Plants 3:1–6. https://doi.org/10.1038/nplants.2017.107

4. ChristeyMC. 1997. Transgenic crop plants using Agrobacterium

rhizogenes mediated transformation, p 99–111. In Doran PM (ed), Hairy

roots: Culture and applications. Harwood Academic Publishers,

Amsterdam.

5. Kiryushkin AS, Ilina EL, Guseva ED, Pawlowski K, Demchenko KN. 2022.

Hairy CRISPR: genome editing in plants using hairy root transformation.

Plants 11:51. https://doi.org/10.3390/plants11010051

6. ThompsonMG, Cruz-Morales P, MooreWM, Pearson AN, KeaslingJD,

SchellerHV, ShihPM. 2020. Draft genome sequence of Agrobacterium

fabrum ARqua1. Microbiol Resour Announc 9:e00506–20.

7. Rodrigues SD, Karimi M, Impens L, Van Lerberge E, Coussens G, Aesaert

S, Rombaut D, Holtappels D, Ibrahim HMM, Van Montagu M, Wagemans

J, Jacobs TB, De Coninck B, Pauwels L. 2021. Ecient CRISPR-mediated

base editing in Agrobacterium Spp. Proc Natl Acad Sci U S A

118:e2013338118. https://doi.org/10.1073/pnas.2013338118

8. Wilson K. 2001. Preparation of genomic DNA from bacteria. Curr Protoc

Mol Biol 56:2–4. https://doi.org/10.1002/0471142727.mb0204s56

9. Chen S, Zhou Y, Chen Y, Gu J. 2018. fastp: an ultra-fast all-in-one FASTQ

preprocessor. Bioinformatics 34:i884–i890. https://doi.org/10.1093/

bioinformatics/bty560

10. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017.

Canu: scalable and accurate long-read assembly via adaptive k-mer

weighting and repeat separation. Genome Res 27:722–736. https://doi.

org/10.1101/gr.215087.116

11. Vaser R, Sović I, Nagarajan N, Šikić M. 2017. Fast and accurate de novo

genome assembly from long uncorrected reads. Genome Res 27:737–

746. https://doi.org/10.1101/gr.214270.116

12. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo

CA, Zeng Q, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated

tool for comprehensive microbial variant detection and genome

assembly improvement. PloS One 9:e112963.

https://doi.org/10.1371/journal.pone.0112963

13. Hunt M, Silva ND, Otto TD, Parkhill J, Keane JA, Harris SR. 2015. Circlator:

automated circularization of genome assemblies using long sequencing

reads. Genome Biol 16:1–10. https://doi.org/10.1186/s13059-015-0849-0

14. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. 2010.

Prodigal: prokaryotic gene recognition and translation initiation site

identication. BMC Bioinf 11:1–11. https://doi.org/10.1186/1471-2105-

11-119

15. Chen N. 2004. Using repeat masker to identify repetitive elements in

genomic sequences. Curr Protoc Bioinf 5:4–10. https://doi.org/10.1002/

0471250953.bi0410s05

16. Nawrocki EP, Eddy SR. 2013. Infernal 1.1: 100-fold faster RNA homology

searches. Bioinformatics 29:2933–2935. https://doi.org/10.1093/

bioinformatics/btt509

17. Lowe TM, Eddy SR. 1997. tRNAscan-SE: a program for improved

detection of transfer RNA genes in genomic sequence. Nucleic Acids Res

25:955–964. https://doi.org/10.1093/nar/25.5.955

18. Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R. 2020.

TBtools: an integrative toolkit developed for interactive analyses of big

biological data. Mol Plant 13:1194–1202. https://doi.org/10.1016/j.molp.

2020.06.009

Announcement Microbiology Resource Announcements

November 2023 Volume 12 Issue 11 10.1128/MRA.00554-23 3

ResearchGate has not been able to resolve any citations for this publication.

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fastp: an ultra-fast all-in-one FASTQ preprocessor

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Long-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. However, given the relatively high error rates of such technologies, efficient and accurate assembly of large repeats and closely related haplotypes remains challenging. We address these issues with Canu, a successor of Celera Assembler that is specifically designed for noisy single-molecule sequences. Canu introduces support for nanopore sequencing, halves depth-of-coverage requirements, and improves assembly continuity while simultaneously reducing runtime by an order of magnitude on large genomes versus Celera Assembler 8.2. These advances result from new overlapping and assembly algorithms, including an adaptive overlapping strategy based on tf-idf weighted MinHash and a sparse assembly graph construction that avoids collapsing diverged repeats and haplotypes. We demonstrate that Canu can reliably assemble complete microbial genomes and near-complete eukaryotic chromosomes using either PacBio or Oxford Nanopore technologies, and achieves a contig NG50 of greater than 21 Mbp on both human and Drosophila melanogaster PacBio datasets. For assembly structures that cannot be linearly represented, Canu provides graph-based assembly outputs in graphical fragment assembly (GFA) format for analysis or integration with complementary phasing and scaffolding techniques. The combination of such highly resolved assembly graphs with long-range scaffolding information promises the complete and automated assembly of complex genomes.

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The assembly of long reads from Pacific Biosciences and Oxford Nanopore Technologies typically requires resource intensive error correction and consensus generation steps to obtain high quality assemblies. We show that the error correction step can be omitted and high quality consensus sequences can be generated efficiently with a SIMD accelerated, partial order alignment based stand-alone consensus module called Racon. Based on tests with PacBio and Oxford Nanopore datasets we show that Racon coupled with Miniasm enables consensus genomes with similar or better quality than state-of-the-art methods while being an order of magnitude faster.

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