# Improvement of the Bos taurus Genome Assembly using New Sequencing Technologies

Camille Eché, Carole Iampietro, Clement Birbes, Andreea Dreau, Claire Kuchly, Arnaud Di Franco, Christophe C. Klopp, Thomas Faraut, Sarah Djebali, Adrien Castinel, et al.

► To cite this version:

Camille Eché, Carole Iampietro, Clement Birbes, Andreea Dreau, Claire Kuchly, et al. Improvement of the Bos taurus Genome Assembly using New Sequencing Technologies. Discoveries Roadshow 2023 - PacBio, PacBio, May 2023, Paris, France. hal-04096124

HAL Id: hal-04096124

https://hal.inrae.fr/hal-04096124

Submitted on 12 May 2023

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Distributed under a Creative Commons Attribution 4.0 International License
# Improvement of the *Bos taurus* Genome Assembly using New Sequencing Technologies

Camille Eche, Carole Lampietro, Clement Birbes, Andreea Dreau, Claire Kuchly, Arnaud Di Franco, Christophe Klopp, Thomas Faraut, Sarah Djebali, Adrien Castinel, Matthias Zytnicki, Erwan Denis, Mekki Boussaha, Cecile Grohs, Didier Boichard, Christine Gaspin, Denis Milan, and Cecile Donnadieu

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GenOToul
GeT

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http://get.genotoul.fr
@Get_Genotoul

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http://bioinfo.genotoul.fr
@BioinfoGenotoul

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GenOToul
Bioinfo

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Acquire advanced expertise on the **new high throughput sequencing technologies available**

- Comparative potentials of technologies

- Identification of combinations of technologies to be implemented according to the objectives

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... in four axes:

**The Genome.** Genome assembly and variant detection

**The Epigenome.** Studies of epigenetic marks that regulate gene expression

**The Metagenomes.** In-depth knowledge of communities

**Data Management.** High molecular weight DNA extraction and evolution of the IT infrastructure

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**Bos taurus**

- 2.7 Gb genome size

- 30 Chromosomes

- 40 % Repeats

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**Zea mays**

- 2.4 Gb genome size

- 10 Chromosomes

- **quasi homozygous**

- 85 % Repeats
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# Why assemble a Charolais breed Bos taurus Genome?

- Leading suckling cattle breed in Europe (25% of the total cows)

- International extension

- Developed on grazing and extensive production systems

- Excellent maternal qualities with high growth potential and an excellent beef

- The specificities of its genome are poorly known

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# The reference genome ARS-UCD1.2

* Submitted by USDA Ars on April 2018

* Line-bred Hereford cow who was selected for her high level of inbreeding.

<table><thead><tr><th></th><th>ARS-UCD1.2</th></tr></thead><tbody><tr><td rowspan="5">Assemblage</td><td>Data type</td><td>CLR</td></tr><tr><td>Quantity</td><td>80X</td></tr><tr><td>Number of contigs</td><td>3 077</td></tr><tr><td>Total size</td><td>2 700 000 000</td></tr><tr><td>N50 contigs length</td><td>12 000 000</td></tr><tr><td rowspan="5">Scaffolding</td><td>Data type</td><td>Hi-C / Optical + Recombination map</td></tr><tr><td>Quantity</td><td>84X Hi-C</td></tr><tr><td>Number of scaffolds</td><td>2 211</td></tr><tr><td>Total size</td><td>2 715 853 794</td></tr><tr><td>N50 scaffolds length</td><td>103 308 737</td></tr><tr><td></td><td>BUSCO</td><td>C:95.8%</td></tr></tbody></table>

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# Technologies used for the study

<table><thead><tr><th>1<br>Sample</th><th>2<br>Sequencing technology</th><th>3<br>Library type</th></tr></thead><tbody><tr><td>Father</td><td>Oxford Nanopore<br>Illumina</td><td>Ligation sequencing gDNA<br>10X Genomics Chromium<br>Hi-C</td></tr><tr><td>Mother</td><td>Oxford Nanopore<br>Illumina<br>PacBio</td><td>Ligation sequencing<br>gDNA<br>10X Genomics Chromium<br>Hi-C<br>Circular Long Read</td></tr><tr><td>Heifer</td><td>Oxford Nanopore<br>Illumina<br>PacBio</td><td>Ligation sequencing gDNA<br>10X Genomics Chromium<br>PCR Free 2x250pb<br>Hi-C<br>Circular Long Read<br>Consensus Long Read</td></tr></tbody></table>

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Create reference datasets for

- Genome assembly

- Haplotyping

- Variability discovery

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# Methods and pipelines used

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Long reads assemblies from ONT and CLR PacBio data

## 10X Chromium assembly

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Phased assembly with HiFi and parental illumina reads

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Phased assembly with HiFi and Hi-C data

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nextflow

https://forgemia.inra.fr/seqoccin/
Assembly metrics

<table><thead><tr><th></th><th>ARS-UCD1.2</th><th>ONT</th><th>HiFi</th></tr></thead><tbody><tr><td>Data type</td><td>CLR</td><td>ONT</td><td>HiFi CCS</td></tr><tr><td>Quantity</td><td>80X</td><td>58X</td><td>40X</td></tr><tr><td>Number of contigs</td><td>3 077</td><td>7 226</td><td>1 444</td></tr><tr><td>Total size</td><td>2 700 000 000</td><td>2 701 288 401</td><td>3 244 632 679</td></tr><tr><td>N50 contigs length</td><td>12 000 000</td><td>23 641 545</td><td>84 059 894</td></tr><tr><td>Data type</td><td>Hi-C / Optical +<br>Recombination map</td><td>Hi-C / 10X</td><td>Hi-C / 10X</td></tr><tr><td>Quantity</td><td>84X Hi-C</td><td>28X / 95X</td><td>28X / 95X</td></tr><tr><td>Number of scaffolds</td><td>2 211</td><td>4 600</td><td>1 391</td></tr><tr><td>Total size</td><td>2 715 853 794</td><td>2 705 347 253</td><td>3 244 660 179</td></tr><tr><td>N50 scaffolds length</td><td>103 308 737</td><td>100 959 810</td><td>87 697 707</td></tr><tr><td>BUSCO</td><td>C:95.8%</td><td>C:95.2%</td><td>C:95.9%</td></tr></tbody></table>

Scaffolding ( after polishing )

HiFi

ONT polished

ONT

BUSCO Assessment Results

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Charolais reference vs ARS reference

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RepeatMasker / RepeatModeler representation of HiFi assembly and
ONT assembly.

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Dgenies Dot-Plot of HiFi Final assembly against Bos taurus reference
ARS-UCD1.2

The additional information in the HiFi assembly is mainly complex duplications.
SeqOccln **Phased assembly**

A diploid assembly is an assembly in which the maternal and paternal haplotypes are separated to create 2 sets of chromosomes.

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Different methods exist to create a diploid assembly:

- 1 Method using distant connection to separate haplotypes: Hi-C

- 1 Method resolving haplotype based on parental K-mers: Yak
Phased assembly

<table><thead><tr><th></th><th>ARS-UCD1.2</th><th>HiFi hifiasm parent hap1</th><th>HiFi hifiasm parent hap2</th><th>HiFi hifiasm Hi-C hap1</th><th>HiFi hifiasm Hi-C hap2</th></tr></thead><tbody><tr><td>Data type</td><td>CLR</td><td>CCS + Trio</td><td>CCS + Trio</td><td>CCS + Hi-C</td><td>CCS + Hi-C</td></tr><tr><td>Quantity</td><td>80X</td><td>40X</td><td>40X</td><td>40X + 28X</td><td>40X + 28X</td></tr><tr><td>Number of contigs</td><td>3 077</td><td>2 871</td><td>2 300</td><td>2 685</td><td>2 136</td></tr><tr><td>Total size</td><td>2 700 000 000</td><td>3 156 028 877</td><td>3 113 483 345</td><td>3 0177 978 241</td><td>3 184 033 110</td></tr><tr><td>N50 contigs length</td><td>12 000 000</td><td>71 619 842</td><td>69 165 538</td><td>80 106 842</td><td>71 644 334</td></tr><tr><td>BUSCO</td><td>C:95.8%</td><td>C:95.8%</td><td>C:95.3%</td><td>C:95.8%</td><td>C:95.7%</td></tr><tr><td>Phasing ratio</td><td>*</td><td>97.3%</td><td>96.7%</td><td>62.6%</td><td>60.5%</td></tr><tr><td>Contigs phasing ratio</td><td>*</td><td>97.5%</td><td>96.9%</td><td>84.6%</td><td>85.6%</td></tr></tbody></table>

Hi-C separation is less efficient than parental separation but works fine on a contig level.
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# Assembly production

229 High quality assemblies generated

## Sus scrofa :

- 5 CLR assemblies

- 4 HiFi assemblies

## Ovis aries :

- 10 CLR assemblies

- 1 HiFi phased assembly

## Bos Taurus :

- 6 ONT assemblies

- 9 CLR assemblies

- 154 CLR assemblies for variant

- 5 HiFi phased assemblies

## Coturnix Japonica :

- 1 HiFi assembly

- 1 ONT assembly

## Capra Hircus :

- 7 CLR assemblies

- 1 HiFi phased assembly

## Zea Mays :

- 25 HiFi assemblies

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# Conclusions and upcoming work

* Improvement of the Bos taurus genome

* ✓ One high quality consensus genome GCA_947034695.1 significantly larger than ARS-UCD1.2 -

* ✓ Two haplotyped trio haplotyped trio high quality reference genome

* ✓ Contribution to the bovine pangenome for the Charolais breed

* Production of whole genome LONG READ sequences for ~150 animals corresponding to several breeds

* ✓ Study structural variations at the whole genome level

* ✓ Construction of several genome assemblies (corresponding to several breeds) -> study the pangenome

* Production of both long and short reads for several trios

* ✓ Construction of genome references using long read data

* ✓ Construction of haplotype graphs using the trio-binning approach

-> Construct several breed specific haplotype graphs

-> study the pangenome

<table><thead><tr><td>Breed</td><td>Number of animals</td></tr></thead><tbody><tr><td>HOL</td><td>25</td></tr><tr><td>MON</td><td>25</td></tr><tr><td>NOR</td><td>25</td></tr><tr><td>BSW</td><td>5</td></tr><tr><td>SIM</td><td>5</td></tr><tr><td>ABO</td><td>10</td></tr><tr><td>TAR</td><td>5</td></tr><tr><td>VOS</td><td>4</td></tr><tr><td>BLA</td><td>10</td></tr><tr><td>CHA</td><td>10</td></tr><tr><td>LIM</td><td>10</td></tr><tr><td>AUB</td><td>10</td></tr><tr><td>FLA</td><td>3</td></tr><tr><td>PAR</td><td>3</td></tr><tr><td>Total</td><td>150</td></tr></tbody></table>

<table><thead><tr><td>Trios</td><td>Breeds</td></tr></thead><tbody><tr><td>Trio 1</td><td>CHA</td></tr><tr><td>Trio 2</td><td>CHA</td></tr><tr><td>Trio 3</td><td>HOL x NMD</td></tr><tr><td>Trio 4</td><td>YAK x MON</td></tr><tr><td>Trio 5</td><td>AUB</td></tr><tr><td>Trio 6</td><td>BAQ</td></tr><tr><td>Trio 7</td><td>ABO</td></tr><tr><td>Trio 8</td><td>TAR</td></tr><tr><td>Trio 9</td><td>VOS</td></tr></tbody></table>
Conclusion

nextflow

forgemia.inra.fr/seqoccin

SCIENTIFIC
DATA

110110
0111101
11011110
01110101

Data paper accepted

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data.gouv.fr

https://entrepot.recherche.data.gouv.fr/dataverse/seqoccin

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https://github.com/GeTPlaGe/SeqOccIn

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https://www.ebi.ac.uk/ena/browser/view/PRJEB60075

Thanks !
# Project partners

SeqOccln

## Coordination

Cécile Donnadieu
Christine Gaspin
Carole Iampietro
Denis Milan

INRAE
la science pour la vie, l'union, la ferme

Liberté • Spécience • Fraternité
RÉPUBLIQUE FRANÇAISE

UNION EUROPÉENNE

l'Europe
s'engage
en Occitanie

Occitanie

<figure><img src="image_41.png" /></figure>

GenOTOL
GeT

<figure><img src="image_42.png" /></figure>

GenOTOL
Bioinfo

## Axe1 Génomique

Clément Birbes
Arnaud Di-Franco
Andreea Dréau
Camille Eché
Thomas Faraut
Carole Iampietro
Christophe Klopp
Claire Kuchly
Camille Marcuzzo
Amandine Suin
Matthias Zytnicki

## GenPhySE

Julie Demars
Cédric Cabau
Sylvie Combes
Patrice Dehais
Thomas Faraut
Katia Feve
Nathalie Iannucelli
Sophie Leroux
Géraldine Pascal
Frédérique Pitel

<figure><img src="image_43.png" /></figure>

GenPhySE

Génétique, Physiologie et Systèmes d'Elevage

## Axe2 Epigénétique

Remy Félix Serres
Paul Terzian
Celine Vandecasteele
Christophe Klopp

## MIAT

Matthias Zytnicki

## Axe3 Métagénomique

Adrien Castinel
Jean Mainguy
Olivier Bouchez
Géraldine Pascal
Claire Hoede

## GABI

Didier Boichard
Mekki Boussaha
Sébastien Fritz
Cécile Grohs
Aurelien Capitan

## Axe 4 Transversal

Amandine Broha
Abdias-Archimede Towe-Patipe
Erwan Denis
Romain Therville
Didier Laborie
Céline Noirot
Gérald Salin
Marie-Stéphane Trotard

## Le Moulon

Alain Charcosset
Johann Joets
Delphine Madur
Stéphane Nicolas
Rémi Séraphin
Clémentine Vitte

<figure><img src="image_44.png" /></figure>

APIS-GENE

Investir Innoyer Valoriser

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AURIVA

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Célitest

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EVOLUTION

X Créalim

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GENES DIFFUSION

Nouvelle ère, new progress

<figure><img src="image_49.png" /></figure>

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Capgenes

ALLIANCE

<figure><img src="image_51.png" /></figure>

Innovation Durable
en Génétique Porcine

AXIOM

l'évidence génétique

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Nucleus

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CHOICE

CNBL

Comité National
Brebis Laitières

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SYSAAF

LALLEMAND

ADISSEO
A DÉBATEUR DE COMPÉTENCE

MA
TOULOUSE

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GABI

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LE MOULON
Génétique
végétale UMR

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biogemma

Limagrain

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euràlis
UNION EUROPÉENNE

FRAGT
2 ans

syngenta

MAISADOUR
semences

CAUSSADE
semences

KWS

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