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A Resource �For Helminth Genomics

APRIL 2, 2024

MATT BERRIMAN �SARAH DYER

Spring Meeting 2024,

University of Liverpool

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Introduce WormBase ParaSite and our latest release
Introduce workshop topics
Workshop part 1

Refreshments

Workshop part 2
Discussion on annotation

A Resource For Helminth Genomics

Programme

workshop materials

https://mberriman.github.io/BSP2024/

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WormBase is an international consortium providing the research community with information concerning the genetics, genomics and biology of Caenorhabditis elegans and related nematodes.

C. elegans

C. brenneri

C. briggsae

C. japonica

C. remanei

Brugia malayi

Onchocerca volvulus

Pristionchus pacificus

Strongyloides ratti

Trichuris muris

…increasing need for a new resource meeting the needs of parasitologists…

A Resource For Helminth Genomics

A Bit of Background…

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parasite.wormbase.org

,

Twitter Followers

Annual Users

Page Views

A Resource For Helminth Genomics

2023

parasite.wormbase.org

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Annual Users

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A Resource For Helminth Genomics

parasite.wormbase.org

Top Accessing Countries

USA

CHINA

UK

GERMANY

INDIA

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69

206

Platyhelminths

Nematodes

A Resource For Helminth Genomics

19

Release

208

Species

275

Genomes

More genomes than ever before!

2024

UPDATES SINCE RELEASE 7 (2016)

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Platyhelminths

Rhabditophora

Tapeworms

Flukes

Monogenea

A Resource For Helminth Genomics

Nematodes

Clade I

Clade C

Clade V

Clade IV

Clade III

2024

More genomes than ever before!

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A Resource For Helminth Genomics

FROM WORMBASE PARASITE RELEASE 7 TO RELEASE 19

Genome Statistics

Contiguity describes how many gaps are in an assembly. In a perfect assembly the number of assembly scaffolds equals the number of chromosomes.

If all of the scaffolds of the assembly are lined up, longest to shortest, half the assembled bases will be in scaffolds ≥ N50. The higher, the better!

Completeness: BUSCO assesses genome completeness by looking for genes that are single-copy and highly conserved across a broad range of eukaryotes. A higher percentage of single BUSCO genes, indicates a more complete dataset.

BUSCO ASSEMBLY, assesses the assembly quality of a genome by predicting a gene-set ab initio using AUGUSTUS.
BUSCO ANNOTATION, assesses annotation completeness by looking for BUSCO genes within existing gene predictions.

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A Resource For Helminth Genomics

More and higher quality assemblies

FROM WORMBASE PARASITE RELEASE 7 TO RELEASE 19

Short-read sequencing only

Long-read sequencing involved

Release 7

Release 19

Assembly updates

Release 7 to Release 19

86

59

145

Log(N50)

Number of Genomes

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A Resource For Helminth Genomics

Improved gene models?

FROM WORMBASE PARASITE RELEASE 7 TO RELEASE 19

Annotation updates

From Release 7 to Release 19

145

Release 7

Release 19

THE OBSERVED ANNOTATION QUALITY IMPROVEMENT DOES NOT MATCH THE BOOST IN ASSEMBLY QUALITY

Long-read sequencing

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Workshop, part one

Overview and aims

Browsing genomes
Browsing gene/transcript pages
Querying WormBase ParaSite with BioMart

A Resource For Helminth Genomics

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What is a gene?

A Resource For Helminth Genomics

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What is a gene?

Gene page

A Resource For Helminth Genomics

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What is a gene?

Gene page

Transcript page

A Resource For Helminth Genomics

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What is a gene?

Gene page

Transcript page

A Resource For Helminth Genomics

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What does the encoded protein do?

A Resource For Helminth Genomics

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Functional Annotation

Gene ontology - a formal representation of knowledge about a gene with respect to three aspects:

Molecular Function
Cellular Component
Biological Process

A Resource For Helminth Genomics

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Functional Annotation

Usually, by knowing which conserved domains exist in a protein, you can make accurate inferences about its function!

“a resource that provides functional analysis of protein sequences by classifying them into families and predicting the presence of domains and important sites..”

InterPro

A Resource For Helminth Genomics

How we do go from a string of amino acids to predicting what this protein might do in the cell? This is where another type of database comes in: protein family databases. For the vast majority of predicted protein sequences, nobody will have done experiments to test what its function is. However, we can use the principle of homology to take proteins that are well-studied in one experimental system and infer that proteins of similar sequence in other organisms are likely to have similar structure, and therefore similar function. In reality, protein sequences are analysed in terms of domains: these are subsequences of a protein that have a defined tertiary structure or sequence motif, conferring a defined function. A protein can consist of several domains. When comparing proteins between organisms, often the region encoding a protein domain is highly conserved whilst the bit that connects different domains together is more divergent.

A well known example of a protein domain database is Pfam. Pfam uses multiple sequence alignments of the known proteins with a certain domain to capture a representative model (a profile Hidden Markov Model) of that domain. Other protein domain databases, that might use slightly different methods to define domains, are: CATH, CDD, HAMAP, MobiDB Lite, Panther, PIRSF, PRINTS, Prosite, SFLD, SMART, SUPERFAMILY and TIGRfams. Luckily for us, all of these databases are united under the InterPro consortium.

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3D Protein Structure

Proteins are 3D molecules, and their 3D structure determines their function

Knowledge of protein's 3D structure is a huge hint for understanding how the protein works

AlphaFold

https://alphafold.ebi.ac.uk/

Artificial Intelligence (AI) system

Computational prediction of protein structures with unprecedented accuracy and speed.

A Resource For Helminth Genomics

Knowledge of protein's 3D structure is a huge hint for understanding how the protein works, and use that information for different purposes; control or modify protein's function, predict what molecules bind to that protein and understand various biological interactions, assist drug discovery or even design our own proteins.

Protein structure prediction is the inference of the three-dimensional structure of a protein from its amino acid sequence—that is, the prediction of its secondary and tertiary structure from primary structure.

Predicting the 3D structure of proteins is one of the fundamental grand challenges in biology. By solving this challenge, we can dramatically deepen our understanding of human health, disease, and our environment, especially within areas like drug design and sustainability.

AlphaFold, the state-of-the-art AI system developed by DeepMind, is able to computationally predict protein structures with unprecedented accuracy and speed. Working in partnership with EMBL’s European Bioinformatics Institute (EMBL-EBI), we’ve released over 200 million protein structure predictions by AlphaFold that are freely and openly available to the global scientific community. Included are nearly all catalogued proteins known to science – with the potential to increase humanity’s understanding of biology by orders of magnitude.

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Why would you want to find orthologues in other species?

If the function of a gene is not known, you can check the function of its orthologue in other species like C. elegans, where direct characterisation is more likely to have occurred.

Often orthologs share many functions/roles.

Comparative Genomics

Orthologues and Paralogues

Further insights into gene function often come from exploring its evolutionary relationship to other genes, both within the same genome, and across different genomes.

A Resource For Helminth Genomics

Another approach to understanding what a gene does is comparing its sequence to other genes, both within the same genome, and across different genomes.

This analysis is called Comparative Genomics

Wait! What orthologues and paralogues are? Read here and/or here to find more!

In a nutshell�Orthologues: Genes which evolved from a common ancestral gene by speciation that usually have retained a similar function in different species.�Paralogues: Paralogs are homologous genes that arise from gene duplication events. Their common ancestry and replicated sequence often leads to similar structure and function in related pathways and protein complexes.�

Why would researchers want to find orthologues in other species for their gene of interest? Function Annotation! If the function of this gene in S. mansoni is not known, you can check the function of its orthologues genes in other species like C. elegans or other Schistosomas. Many times you will find that orthologous genes function will be similar to this of your gene of interest.��

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Efficiently query WormBase ParaSite using BioMart

A Resource For Helminth Genomics

Echinococcus multilocularis	CDKD1.14
Echinococcus multilocularis	CLK2.7
Echinococcus multilocularis	EmuJ_000000300
Echinococcus multilocularis	EmuJ_000000800
Echinococcus multilocularis	EmuJ_000001200
Echinococcus multilocularis	EmuJ_000001400
Echinococcus multilocularis	EmuJ_000002000
Echinococcus multilocularis	EmuJ_000002800
Echinococcus multilocularis	EmuJ_000003100
Echinococcus multilocularis	EmuJ_000003600

GENE ONTOLOGIES

ALPHAFOLD

PROTEIN DOMAINS

ORTHOLOGUES

PARALOGUES

MULTI-GENE OUTPUT

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https://mberriman.github.io/BSP2024/

A Resource For Helminth Genomics

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Part2

6. Overview and Aims

7. Tools

BLAST

EXERCISE

The genome browser

VEP

EXERCISE

8. The WormBase ParaSite Expression browser

EXERCISE

9. Gene-set enrichment analysis

EXERCISE

A Resource For Helminth Genomics

This figure represents the steps that are needed to transform information encoded in the DNA into a polypeptide and eventually a functional protein. The starting information is encoded in the genome. A gene encodes, among other things, the transcription start and transcription end. These are the nucleotides from where an RNA copy of the DNA will be generated. UTRs. This is the gene’s transcript + multiple transcripts This copy is the pre-mRNA which is formed by exons and introns. Maturation of the mRNA molecule happens as it is transcribed and involves the splicing (removal) of introns with the concomitant joining of exons, addition of a CAP at the 5’ end and a polyadenylation tail (many As - AAAAAAA) at the 3’end. A processed mRNA will be the template for the translation of the mRNA message into a protein by the ribosome.

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BLAST

CGGAGCGCGTGGC

TACGGCCCGGAAT

GCGGTTAATTGCGGC

TACGGCCCGGAAT

CGGAGCGCGTGGC

TACGGCCCGGAAT

GCGGTTAATTGCGGC

TACGGCCCGGAAT

BLAST compares nucleotide or protein sequences to all sequences of WormBase ParaSite using local alignments

Gene	Species	E-Score
Gene A	S. haematobium	4.3e-63
Gene B	S. haematobium	1e-20
Gene C	S. bovis	0.05
….

calculates an Expectation Score - number of hits expected to be seen by chance, when searching an equivalent sized database.

?

A Resource For Helminth Genomics

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Genome Browsers

Visualise a gene model in its genomic context
Assess gene model correct
Visualise functional genomics data

A Resource For Helminth Genomics

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A Resource For Helminth Genomics

Visualising variants on AlphaFold models

FROM A VCF FILE WITH VARIANTS…

…TO PREDICTING THEIR EFFECT…

…TO VISUALISING THEM ON �3D PROTEIN MODELS…

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Explore, use and analyse transcriptomics data

A Resource For Helminth Genomics

VISUALISE RNA-SEQ DATASETS BY CONDITION

EXPLORE DIFFERENTIAL GENE EXPRESSION EXPERIMENTS

PERFORM GENE-SET ENRICHMENT ANALYSIS

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Gene-set enrichment analysis

A Resource For Helminth Genomics

A METHOD USED TO DETERMINE WHICH BIOLOGICAL PROCESSES, MOLECULAR FUNCTIONS, AND CELLULAR COMPONENTS ARE SIGNIFICANTLY ASSOCIATED WITH A SET OF GENES OR PROTEINS.

FROM A LIST OF GENES…

…TO A LIST OF ENRICHED TERMS OF GENE FUNCTION.

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Show your support!

A Resource For Helminth Genomics

SUBSCRIBE TO OUR MAILING LIST

FOLLOW US ON TWITTER

HELPDESK

@WBParaSite

parasite-help@wormbase.org

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John Tate�Ensembl Web Back-end Project Leader

EMBL's European Bioinformatics Institute �(EMBL-EBI)

Prof. Matthew Berriman�PI

School of Infection & Immunity,

College of Medical,Veterinary & Life Sciences,

University of Glasgow

Dr. Sarah Dyer�PI

EMBL's European Bioinformatics Institute (EMBL-EBI)

Steph Brown�Bioinformatician

School of Infection & Immunity,

College of Medical,Veterinary & Life Sciences,

University of Glasgow

Mehrnaz Charkhchi

Ensembl Web Back-end Developer

EMBL's European Bioinformatics Institute �(EMBL-EBI)

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A Resource For Helminth Genomics

How do we currently annotate?

A FOCUS ON ANNOTATION

Coordinates of each exon, translated vs untranslated regions, ncRNAs
WormBase receives gene predictions from external genome projects
Large-scale producers & small-scale independent researchers
Quality varies

BRAKER PIPELINE

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A Resource For Helminth Genomics

How do we currently annotate?

MANUAL REVIEW IS ALWAYS NECESSARY BUT RARELY AVAILABLE

WHAT'S WRONG WITH THE 5' END OF THIS GENE?

SOLUTION

SPLIT FIRST INTRON TO MAKE A NEW 5' GENE?

APPEARS TO BE TWO QUITE DIFFERENT RNA-SEQ COVERAGE LEVELS

...INDEPENDENT START SITES

Steve Doyle

APOLLO ANNOTATION TOOL

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A Resource For Helminth Genomics

How do we currently annotate?

MANUAL REVIEW IS ALWAYS NECESSARY BUT RARELY AVAILABLE

...AND THE SPLIT IS SUPPORTED BY RNASEQ EVIDENCE

Z

W

APOLLO ANNOTATION TOOL

ARTEMIS COMPARISON TOOL

THE W LOCUS IS ALREADY ANNOTATED AS TWO GENES...

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A Resource For Helminth Genomics

How do we currently annotate?

MANUAL REVIEW IS ALWAYS NECESSARY BUT RARELY AVAILABLE

APOLLO ANNOTATION TOOL

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A Resource For Helminth Genomics

How do we currently annotate?

MANUAL REVIEW IS ALWAYS NECESSARY BUT RARELY AVAILABLE

APOLLO ANNOTATION TOOL

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A Resource For Helminth Genomics

How do we currently annotate?

MANUAL REVIEW IS ALWAYS NECESSARY BUT RARELY AVAILABLE

APOLLO ANNOTATION TOOL

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A Resource For Helminth Genomics

Description of gene function

AN AREA THAT STILL NEEDS CURATION

FUNCTIONAL ANNOTATION

Importing descriptions from UniProt

Historically pushing annotations to UniProt
Uniprot and WBPS out of sync sometimes

Gene names from the community imported as synonyms

Reactively not systematically

Coming soon: Automated gene descriptions from WB for WB species

GENE SYNONYM IMPORTED FOR STRONGYLOIDES STERCORALIS

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A Resource For Helminth Genomics

A FOCUS ON ANNOTATION

Community annotation

DO I WANT TO GET INVOLVED?

HOW MUCH TIME COULD I SPEND ON IT?

WHAT WOULD I WANT TO GET OUT OF IT?

WHAT SHOULD BE THE FOCUS?

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User comments on gene pages

A Resource For Helminth Genomics

A FORM OF COMMUNITY ANNOTATION

Gene Name

Gene Structure

Gene Description