Elateridae Ortholog Target Enrichment Baits

The Bait-100-85_Elateridae.fas bait set contains 40190 100nt baits designed from 8 Elateroidea species targeting 2260 orthologs.

Acknowledgements

Developed under the direction of Jeremy Dettman as part of BioMob WP2

This bait set was made possible by the significant contributions from the following scientists:

• Hermes Escalona, Adam Ślipiński, (Australian National Insect Collection, CSIRO, Australia), and Duane McKenna (University of Memphis, USA) for providing the transcriptomes of Melanotus villosus and Drilus concolor

• Dr. Hume Douglas and Dr. Adam Brunke (Agriculture & Agri-Food Canada) for their instrumental guidance on the design of the bait set

• Brian Brunelle (Arbor BioSciences) for probe creation and QA/QC prior to manufacturing

• Dr. Jeremy Dettman, Julie Chapados, Robin Richter, (Agriculture & Agri-Food Canada) for the extensive validation and implementing of the bait set

Files

• Bait-100-85_Elateridae.fas: Final bait set with OrthoDB v10 headers
• Bait-100-85_Elateridae.fas.fasta: Final bait set with Phyluce compatible UCE headers
• Elateridae_Submission.fasta: Final file generated by elateridae_baits.py submitted to Arbor BioSciences for bait design

Design Methodology

• Gathered 4296 Orthologs from OrthoDB v10 for Coleoptera (Present in 80% of Species, Single Copy in All Species)

wget https://www.orthodb.org/tab?query=&level=7041&species=7041&universal=0.8&singlecopy=1&limit=5000 > OrthoDB_Coleoptera_80_Percent.tab

• Prepare file for Orthograph

awk 'BEGIN {FS="\t"; OFS="\t"} {print $1, $6, $5}' OrthoDB_Coleoptera_80_Percent.tab > OrthoDB_Coleoptera_cut.tsv

2. Retrieve Official Gene Sets for the all Coleoptera species odb10_arthropoda_fasta.tar.gz

   OrthoDB Species Identifier	Species Name
   110193_0	Nicrophorus vespilloides
   116153_0	Aethina tumida
   1629725_0	Oryctes borbonicus
   166361_0	Onthophagus taurus
   217634_0	Anoplophora glabripennis
   224129_0	Agrilus planipennis
   7070_0	Tribolium castaneum
   7539_0	Leptinotarsa decemlineata
   77166_0	Dendroctonus ponderosae

4. Orthograph was prepared following the instructions outlined in the Orthograph README by adding the 9 OGS and the OrthoDB_Coleoptera_cut.tsv
5. Orthograph was run on the following Elateroidae transcriptomes assembled using rnaSPAdes v 1.13 after adaptor trimming with BBDuk

• I189 Melanotus villosus
• I246 Drilus concolor
• SRR4413771 Chauliognathus flavipes
• SRR7300147 Pyrearinus fragilis
• SRR7300148 Pyrearinus fragilis
• SRR7300149 Monocrepidius
• SRR6286833 Melanotus cribricollis
• SRR6345452 Photinus pyralis

6. Orthograph results were merged using Orthograph-reporter
7. Amino acid orthologs were aligned using T_Coffee 11.0.8 additionally outputting score_ascii files
8. Protein alignments were converted to nucleotide alignments using Tranalign from EMBOSS 6.6.0 resulting in 4264 nucleotide alignments
9. elateridae_baits.py performed the following steps:
   • Scanned each score_ascii file with sliding window starting with a length of 200 AA. Each alignment position has a TSC score from 1-9 so a max score of 200 AA is 1800. The window with a sum above 95% of the max TCS score was considered a conserved block.
   • 2764 Corresponding conserved blocks were excised from the nucleotide transalignments by multiplying the start and stop indices of protein alignments by 3
   • Any sequences with at least 20% gapped bases were removed from conserved blocks
   • 2260 Conserved blocks were found containing a minimum of 5 of the 8 Elateroidea species
   • Curated blocks were created by randomly selecting 3 Elateroidea specimens
   • Curated blocks were appended into a multi-fasta Elateridae_Submission.fasta and submitted to Arbor Biosciences for MyBaits manufacturing

Arbor BioSciences Bait Design

The following procedures were performed by Brian Brunelle, Arbor Biosciences

1. 6780 loci provided for bait design (Ns were replaced with Ts if they occurred consecutively for 1-10 bases)
2. Using RepeatMasker, soft-masked the input sequences for simple repeats and repeats found in the Elateridae database; 0.1% masked (all simple repeats)
3. 1.15X Tiling (100 nt bait from every 85 nt) interval based on GC, ΔG,
4. Each bait candidate was BLASTed against the 25 provided genomes;

Species	TaxID	Common Name	Accession
Oryzaephilus surinamensis	41112	saw-toothed grain beetle	GCA_004796505.1
Popillia japonica	7064	Japanese beetle	GCA_004785975.1
Asbolus verrucosus	1661398	a darkling beetle	GCA_004193795.1
Protaetia brevitarsis	348688	a scarab beetle	GCA_004143645.1
Diabrotica virgifera	50389	western corn rootworm	GCF_003013835.1
Harmonia axyridis	115357	a lady beetle	GCA_003865315.1
Harmonia axyridis	115357	a lady beetle	GCA_003865295.1
Harmonia axyridis	115357	a lady beetle	GCA_003865275.1
Coccinella septempunctata	41139	seven-spotted ladybird	GCA_003568925.1
Harmonia axyridis	115357	a lady beetle	GCA_003402655.1
Aleochara bilineata	135921	a rove beetle	GCA_003054995.1
Sitophilus oryzae	7048	rice weevil	GCA_002938485.1
Agrilus planipennis	224129	emerald ash borer	GCF_000699045.2
Anoplophora glabripennis	217634	Asian longhorned beetle	GCF_000390285.2
Leptinotarsa decemlineata	7539	Colorado potato beetle	GCF_000500325.1
Onthophagus taurus	166361	a scarab beetle	GCF_000648695.1
Pogonus chalceus	235516	a ground beetle	GCA_002278615.1
Aethina tumida	116153	small hive beetle	GCF_001937115.1
Tribolium castaneum	7070	red flour beetle	GCF_000002335.3
Oryctes borbonicus	1629725	a scarab beetle	GCA_001443705.1
Dendroctonus ponderosae	77166	mountain pine beetle	GCF_000355655.1
Nicrophorus vespilloides	110193	carrion beetle	GCF_001412225.1
Priacma serrata	50550	a reticulated beetle	GCA_000281835.1
Hypothenemus hampei	57062	coffee berry borer	GCA_001012855.1
Dendroctonus ponderosae	77166	mountain pine beetle	GCA_000346045.2

A hybridization melting temperature (Tm, defined as temperature at which 50% of molecules are hybridized) was estimated for each hit assuming standard myBaits® buffers and conditions.

5. For each bait candidate, one BLAST hit with the highest Tm is first discarded from the results (allowing for 1 hit in the genome), and only the top 500 hits (by bit score) are considered. Based on the distribution of remaining calculated Tm's, we filtered out non-specific baits using the following criteria:

   • Stringent (only specific baits pass). Bait candidates pass if they satisfy one of these conditions:
     • No hits with Tm above 60°C
     • At most 2 hits 62.5 – 65°C
     • At most 10 hits 62.5 – 65°C and at least 1 failing flanking bait
     • At most 10 hits 62.5 – 65°C, 2 hits 65 – 67.5°C, and fewer than 2 passing flanking baits
     • At most 2 hits 62.5 – 65°C, 1 hit 65 – 67.5°C, 1 hit 70°C or above, and < 2 passing flanking baits
   • Moderate (some non-specific baits pass)
     • Additional candidates pass if they have at most 10 hits 62.5 – 65°C and 2 hits above 65°C, and fewer than 2 passing baits on each flank.
   • Relaxed (more non-specific baits pass)
     • Additional candidates pass if they have at most 10 hits 62.5 – 65°C and 4 hits above 65°C, and fewer than 2 passing baits on each flank

6. Optimal bait design, keep only baits that passed “Stringent” BLAST filtering for all 25 genomes and were ≤25% Repeat Masked

7. Final bait set, Bait-100-85_Pass_Filtering_Renamed.fas: 100 nt baits after recommended filtration = 40160 baits • target_sequences.masked.fas = the 6,780 total target sequences soft masked these for simple repeats (0.1% masked) • Bait-100-100.fas = Designed 100 nt baits every 100 nt; 40,471 baits • filter-files = folder of all BLAST analysis from all 25 genomes • Filtered_Baits.fasta = design after keeping only baits that were <=25% soft masked, matched a region of the genome that was <= 25% masked, and passed our Stringent analysis; 34,497 baits

Phyluce Prediction of Bait Effectiveness

In order to provide a rough idea effectiveness, the bait set was compared against 23 Coleoptera assemblies available as of April 2019. Bait set fasta headers were converted to Phyluce "UCE" format and processed via Phyluce, outlined in Tutorial IV: Identifying UCE Loci and Designing Baits To Target Them: In-silico test of the bait design

Table 1. Target Hits Among NCBI Coleoptera Assemblies

Built With

• Python - Programming language
• Conda - Package, dependency and environment management
• Phyluce - Target enrichment data analysis
• BioPython - Tools for biological computation
• Orthograph - Orthology prediction
• OrthoDB- Hierarchical catalog of orthologs
• T_Coffee Multiple sequence alignment
• Tranalign Nucleotide alignments from protein alignments

Contact

Jackson Eyres, Bioinformatics Programmer, Agriculture & Agri-Food Canada
[email protected]

Cite Us

If you use this bait set, please cite the following paper

Douglas, H.B.; Kundrata, R.; Brunke, A.J.; Escalona, H.E.; Chapados, J.T.; Eyres, J.; Richter, R.; Savard, K.; Slipinski, A.; McKenna, D.; Dettman, J.R. Anchored Phylogenomics, Evolution and Systematics of Elateridae: Are All Bioluminescent Elateroidea Derived Click Beetles? Biology 2021, 10, 451. https://doi.org/10.3390/biology10060451

Copyright

Agriculture & Agri-Food Canada, Government of Canada

License

This project is licensed under the MIT License - see the LICENSE file for details

Citations

1. Brunke, A. J., Hansen, A. K., Salnitska, M., Kypke, J. L., Escalona, H., Chapados, J.T., Eyres, J., Richter, R., Smetana, A., Ślipiński, A., Zwick, A., Hájek, J., Leschen, R., Solodovnikov, A. and Dettman, J.R. The limits of Quediini at last (Coleoptera: Staphylinidae: Staphylininae): a rove beetle mega-radiation resolved by comprehensive sampling and anchored phylogenomics. Systematic Entomology. 46: 396-421.

2. Faircloth, B.C. 2016. PHYLUCE is a software package for the analysis of conserved genomic loci. Bioinformatics 32:786-788. doi:10.1093/bioinformatics/btv646.

3. OrthoDB v10: sampling the diversity of animal, plant, fungal, protist, bacterial and viral genomes for evolutionary and functional annotations of orthologs Kriventseva EK et al, NAR, Nov 2018, doi:10.1093/nar/gky1053. PMID:30395283

4. Petersen, M., Meusemann, K., Donath, A. et al. Orthograph: a versatile tool for mapping coding nucleotide sequences to clusters of orthologous genes. BMC Bioinformatics 18, 111 (2017). https://doi.org/10.1186/s12859-017-1529-8