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# A Bos taurus sequencing methods benchmark for assembly, haplotyping, and variant calling

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

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## To cite this version:

Camille Eché, Carole Iampietro, Clément Birbes, Andreea Dréau, Claire Kuchly, et al. A Bos taurus sequencing methods benchmark for assembly, haplotyping, and variant calling. Plant and Genome San Diego 2024, Jan 2024, San Diego, United States. 10, 2024. [https://doi.org/10.5281/zenodo.1176283](https://doi.org/10.5281/zenodo.1176283)

HAL Id: hal-04432803

https://hal.science/hal-04432803v1

Submitted on 26 Feb 2024

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Distributed under a Creative Commons Attribution 4.0 International License
Camille Eché <fnref n="1" />, Carole Iampietro <fnref n="1" />, Clément Birbes ², Andreea Dréau ², Claire Kuchly <fnref n="1" />, Arnaud Di Franco ², Christophe Klopp ², Thomas Faraut ³, Sarah Djebali ³, Adrien Castinel <fnref n="1" />, Matthias Zytnicki ⁴, Erwan Denis <fnref n="1" />, Mekki Boussaha ⁵, Cécile Grohs ⁵, Didier Boichard ⁵, Christine Gaspin ², Denis Milan <fnref n="1" />, Cécile Donnadieu <fnref n="1" />

## Background and objectives

Inspired by the production of reference datasets in the Genome in a Bottle project, we sequenced a Charolais heifer using different technologies: Illumina paired-end, Oxford Nanopore, Pacific Biosciences (HiFi and CLR), 10X Genomics linked-reads, and Hi-C. To generate haplotypic assemblies, we also sequenced both parents with short reads. From these data, we constructed two haplotyped trio high quality reference genomes and a consensus assembly, using current software packages. **The assemblies generated using PacBio HiFi reach a size of 3.2 Gb,** which is significantly larger than the 2.7 Gb ARS-UCD1.2 reference. The BUSCO score of the consensus assembly reaches a completeness of 95.8% completeness among highly conserved mammalian genes. **We also identified 35,866 structural variants greater than 50 base pairs.** This assembly is a contribution to the bovine pangenome for the Charolais breed. These datasets will prove to be useful, allowing the community to gain additional insight into sequencing technologies for applications such as SNP, indel or structural variant calling, and de novo assembly.

More information in our data paper → https://doi.org/10.1038/s41597-023-02249-1

## Sequencing technologies and pipelines used for the bovine trio

<figure><img src="image_4.png" /><figcaption>**Figure 1: Technologies used for the study**</figcaption></figure>

The parents and the heifer were sequenced using Oxford Nanopore Technologies on GridION and PromethION, Chromium 10X and the Hi-C method on MiSeq, HiSeq or NovaSeq6000. The heifer was additionally sequenced using Illumina 2x250pb on NovaSeq6000 and PacBio Sequel II (CLR and CCS (i.e. HiFi reads) mode). For the Trio approach, parent reads (2x150pb) from 10X Genomics Chromium data were used.

<figure><img src="image_5.png" /><figcaption>**Figure 2: Details of the 5 pipelines used to produce assemblies**</figcaption></figure>

a-Long reads assemblies from Oxford Nanopore Technologies and Pacific Biosciences followed by a polishing step for erroneous assemblies and scaffolding step. **b-10X Chromium assembly and scaffolding with Supernova.** **c-Phased assembly with HiFi and parental Illumina reads.** **d-Phased assembly with HiFi and Hi-C data.**

## Comparison of heifer consensus and haplotyped assemblies

<figure><img src="image_6.png" /><figcaption>**Figure 3: Heifer NG(X)% comparison**</figcaption></figure>

Comparison and evolution of the heifer assemblies size metrics after different steps in different assemblies pipelines.

<figure><img src="image_7.png" /><figcaption>**Table 1: Summary of heifer phased produced assemblies**</figcaption></figure>

Details of pipeline used in this study are shown in Figure 2. CLR (80X), CCS (40X) and Hi-C (28X) Illumina (30X)* BUSCO analysis was performed on polished contigs. ** Reference is not haplotyped

## Structural variation and SNPs

<figure><img src="image_8.png" /><figcaption>**Figure 4: Structural variant length distribution**</figcaption></figure>

Length distribution of DEL (left) and INS (right) variants, detected by the different sequencing technologies on the Charolais heifer. Sharp peaks correspond to the structural variability generated by transposable elements (SINE, LTR and LINE). All the long reads technologies exhibit roughly the same distribution underlining the fact that they are comparable in their ability to detect DEL and INS variants (see also Fig 5). This holds also with short reads for DEL variants. In contrast, there is a significant difference between long and short read technologies in their ability to detect INS variants, short reads failing to detect a large proportion of INS variants, the larger the size the more pronounced the trend. This is an expected consequence of the limited size of reads that prevents to recover long insertions. **Long read technology seems imperative for an accurate detection of the full spectrum of structural variants.**

<figure><img src="image_9.png" /><figcaption>**Figure 5: Upset plot of the identified deletions and insertions**</figcaption></figure>

The majority of variants are identified by all the long read technologies as well as for the assembly comparison based variant detection. This upset plot again highlights the added value of long reads for structural variant detection.

**High-quality phased and consensus genome assemblies for Charolais heifer with PacBio, Hi-C and parents data ! Very promising SV detection with long reads data !**

<footnote n="1">¹ INRAE, US 1426, GeT-PlaGe, Genotoul, Castanet-Tolosan, France. ² Plateforme Bio-informatique Genotoul, Mathématiques et Informatique Appliquées de Toulouse, INRAE, Castanet-Tolosan, France. ³ GenPhySE, Université de Toulouse, INRA, INPT, ENVT, Castanet-Tolosan Cedex, F-31326, France. ⁴ Université Fédérale de Toulouse, INRAE, MIAT, 31326, Castanet-Tolosan, France. ⁵ Université Paris-Saclay, INRAE, AgroParisTech, GABI, 78350 Jouy-en-Josas, France.</footnote>