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# Variations de structure et longues lectures

Thomas Faraut

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Thomas Faraut. Variations de structure et longues lectures. Colloque INRAE Genomics, INRAE Genomics, May 2022, Orléans, France. hal-04220082

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Submitted on 27 Sep 2023

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Variations de structure et longues lectures

Colloque INRAE Genomics

Thomas Faraut

Orléans, 17 mai 2022
* Landscape of structural variations with short and long reads

What is the relative contribution of insertions and deletions to the structural variability of genomes ?

* The case of indels

* The case of large insertions and deletions

From short reads to long reads and back

* The variation graph
<figure><img src="image_2.png" /></figure>

SV: the case of short reads

1000 goats (Vargoats project)

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

* Deletions clearly outnumber insertions

<figure><img src="image_6.png" /></figure>

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Project enhanced by the funds donated in the framework of the International Research Fund for Scientific Research
<figure><img src="image_11.png" /></figure>

SV: the case of short reads

Structural variation signatures

<figure><img src="image_12.png" /></figure>

RD: Read depth

DP : Discordant pairs

SR : Split reads

van Belzen et al. npj Precis Onc 5 (2021). https://doi.org/10.1038/s41698-021-00155-6
## Trio2 heifer

<figure><img src="image_13.png" /></figure>

* Symmetry of the size distribution of insertion/deletions is a sign of good health of the detected variants
<figure><img src="image_14.png" /></figure>

# The symmetry of insertions and deletions

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

* Structural variation type INS and DEL are somehow ill-defined

* For small insertions/deletions the term indel preserves this ambiguity
<figure><img src="image_17.png" /></figure>

The symmetry of insertions and deletions

Reference

Individual

<figure><img src="image_18.png" /></figure>

INS

DEL

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

* When comparing two genomes we expect to see a symmetric pattern, the same number of INS and of DEL
Indels detection

AGGCTCCTCCGTCCATGCGATT--GCAGGCC

11111111111111111111111111111111

AGTC---TCCGTCCATGGGATTTGCAGGCC

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

* Indels are detected directly from alignments
<figure><img src="image_23.png" /></figure>

# Indel size distribution: PacBio

## Trio2 heifer

<figure><img src="image_24.png" /></figure>

* With PacBio HiFi, the distribution of indel sizes exhibit a clear symmetric pattern
Indel INS/DEL bias

PacBio HiFi

<figure><img src="image_25.png" /></figure>

Illumina

<figure><img src="image_26.png" /></figure>

$$bias = \frac{n_d - n_i}{n_d + n_i}$$

* PacBio HiFi seems unbiased, while a clear bias is observed for Illumina
Ancestral allele

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

* The identification of the ancestral state is mandatory to identify the mutation, deletion of insertion for a given variant
<figure><img src="image_29.png" /></figure>

# Indels ancestral allele

## Heifer

Variation

<figure><img src="image_30.png" /></figure>

Mutation

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

* Short deletions clearly outnumber short insertions
<figure><img src="image_33.png" /></figure>

# Indels at the population level

## 1000 Bull genomes project

<figure><img src="image_34.png" /><figcaption>Mutation</figcaption></figure>

<figure><img src="image_35.png" /></figure>

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* A large deletion bias and the symmetry is lost
## Vargoats project

1000 goats

<figure><img src="image_37.png" /></figure>

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Decrease of the ins/del with an increasing population size
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GenoTOUL
GeT

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

GenoTOUL
BioInfo

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>
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DEL INS

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$$S_n = \theta \sum_{i=0}^{n} \frac{1}{i}$$
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Affine gap costs

Bwa mem (Smith-Waterman 1981)

Minimap2 (Gotoh 1990)

$$G_c = \alpha + \beta(k - 1)$$

$$G_c = \min\{q + |I|, \tilde{q} + |\tilde{I}|\}$$
* Population data provides information on the dominant mutational mechanism for indel

* For small insertion/deletions (indels), deletions are about two times more frequent than insertions (also documented in the literature)

* Ancestral state reconstruction provides information of the specific mutation for each variant
OTEDOR

<figure><img src="image_52.png" /><figcaption>CLR</figcaption></figure>

<figure><img src="image_53.png" /></figure>

* Illumina fails to detect a large proportion of insertions
OTEDOR

<figure><img src="image_54.png" /><figcaption>Mutation</figcaption></figure>

<figure><img src="image_55.png" /></figure>

<figure><img src="image_56.png" /></figure>
100 SeqOccln CLR bulls

Variation

<figure><img src="image_57.png" /></figure>

Mutation

<figure><img src="image_58.png" /></figure>

<figure><img src="image_59.png" /></figure>
100 SeqOccIn CLR bulls

<figure><img src="image_60.png" /></figure>

<figure><img src="image_61.png" /></figure>

<figure><img src="image_62.png" /></figure>

Increase of the ins/del ratio with the number of samples
SV summary

* In contrast to indels, for medium to large variations, insertion seems to be the predominant mutation mechanism

* A junction is needed between indel and SV catalogues to confirm this potential switch
* All technologies exhibit different kind of bias

* This is especially true for short reads in the context of structural variant detection

* A major source of bias is the use of a single reference genome
<figure><img src="image_63.png" /></figure>

Reference bias

Giab son (HG002)

Reference alleles map better

<figure><img src="image_64.png" /></figure>

* Fraction of alternate allele when aligned to the variation graph (red) with vg or to the reference (blue for bwa, or green with vg)

Garrison et al. Nat Biot 36:875 (2018). https://doi.org/10.1038/nbt.4227
Variation graph

a

<figure><img src="image_65.png" /></figure>

b

<figure><img src="image_66.png" /></figure>

Goodbye reference, hello genome graphs Nat Biotechnol 37, 866–868 (2019)
https://doi.org/10.1038/s41587-019-0199-7
<figure><img src="image_67.png" /></figure>

* Variants detected in 100 bulls (CLR) are used to construct a variation graph

* Illumina data from these same 100 bulls sample are used to genotype them on the variation graph
* For small insertion/deletions (indels), deletions are about two times more frequent than insertions

* In contrast to indels, for medium to large variations, insertion seems to be the predominant mutation mechanism

* Goodbye reference, hello genome graphs
Chronicle of a Disparition Foretold
<figure><img src="image_68.png" /></figure>

BovA2 family

<figure><img src="image_69.png" /></figure>

* There is a sharp peak at 143bp made of insertions and deletions

* This is approximately half the size of a BovA2 SINE
The 143bp peak

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>

<figure><img src="image_72.png" /></figure>

<figure><img src="image_73.png" /></figure>
BovA2 recombination

<figure><img src="image_74.png" /></figure>

<figure><img src="image_75.png" /></figure>

* The high similarity between the two copies can lead to unequal crossing-over
BovA2 recombination

<figure><img src="image_76.png" /></figure>

<figure><img src="image_77.png" /></figure>

<table><thead><tr><th>deletion</th><th>Reference</th><th>Alternate</th></tr></thead><tbody><tr><td>INS</td><td>89%</td><td>85%</td></tr><tr><td>DEL</td><td>79%</td><td>72%</td></tr></tbody></table>

<figure><img src="image_78.png" /></figure>

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BovA2 recombination

<figure><img src="image_84.png" /><figcaption>Product 2</figcaption></figure>

<figure><img src="image_85.png" /></figure>

<table><thead><tr><th>insertion</th><th>Reference</th><th>Alternate</th></tr></thead><tbody><tr><td>INS</td><td>50%</td><td>54%</td></tr><tr><td>DEL</td><td>63%</td><td>73%</td></tr></tbody></table>

<figure><img src="image_86.png" /></figure>

<figure><img src="image_87.png" /></figure>

<figure><img src="image_88.png" /></figure>

<figure><img src="image_89.png" /></figure>
* BovA2 copies in the bovine (ruminants) genome experience an intensive erosion mechanism due to recombination
<figure><img src="image_90.png" /><figcaption>cow</figcaption></figure>

<figure><img src="image_91.png" /><figcaption>Pig</figcaption></figure>

<figure><img src="image_92.png" /><figcaption>Maize</figcaption></figure>

<figure><img src="image_93.png" /></figure>
# Remerciements

SeqOccln

Coordination

Cécile Donnadieu
Christine Gaspin
Carole Lampietro
Denis Milan

<figure><img src="image_94.png" /></figure>

<figure><img src="image_95.png" /></figure>

<figure><img src="image_96.png" /></figure>

Plus spécifiquement impliqués dans les travaux présentés aujourd'hui

Quentin Boone
Mekki Boussaha
Mathieu Charles
Arnaud Di Franco
Thomas Faraut
Christophe Klopp

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APIS-GENE

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AURIVA

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X EVOLUTION

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C-ALTOST

ALLIANCE
REDD

AXOM

CHOICE

CNBL

LAZEMAND

Jicometrics

AVI
biogamma

Linagene

eurális

INRAGT

X Créalisme

X GESTORRISATION

<figure><img src="image_105.png" /></figure>

Capugnos

<figure><img src="image_106.png" /></figure>

NUCLEUS

<figure><img src="image_107.png" /></figure>

Adisseo

syngenta

NARADOUR

Cultivare

<figure><img src="image_108.png" /></figure>