# Workflows for Rapid Functional Annotation of Diverse Arthropod Genomes
## 2 Materials and Methods
### Research Design and Method: Comparing Functional Annotation across Multiple Species  
To test the usefulness of the functional annotation workflows, we selected a set of arthropod genomes (Table 3) with varying assembly quality and state of manual curation. This data set included several well-studied arthropod genomes, such as _Drosophila melanogaster_, _Apis mellifera_, and _Tribolium castaneum_, for comparison. BUSCO [26; 28] version 5.1.2 was used with the protein option and arthropoda_odb10.2019-11-20 database with 1013 markers to analyze all protein sets for completeness (Table 4).  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Species** & \begin{tabular}{c} **Genome** \\ **Assembly** \\ **Accession** \\ \end{tabular} & \begin{tabular}{c} **Genome** \\ **Assembly** \\ **Name** \\ \end{tabular} & \begin{tabular}{c} **Config** \\ **N50** \\ \end{tabular} & \
\begin{tabular}{c} **Scaffold** \\ **N50** \\ \end{tabular} & **Annotation Name** & **Proteins** &
\begin{tabular}{c} **Proteins** \\ **Assigned** \\ **GO Terms** \\ \end{tabular} & **Source** \\ \hline _Apis mellifera_ & & & & & & & & \\ (honey bee) & GCA\_00002195.1 & Amel\_4.5 & 5,832,476 & 13,619,445 & OGSv\_3.3 & 15,314 & 39.91\% & [29] \\ _Drosophila melanogaster_ & & & & & & & \\ (fruit fly) & & & & & & & & \\ _Tribolium castaneum_ & & & & & & & \\ (red flour beetle) & GCA\_00002335.3 & TCAS\_5.2 & 73,049 & 4,456,720 & TCAS\_OGS\_v3 & 18,534 & 44.98\% & [31] \\ _Larotckens heersers_ & & & & & & & & \\ (Western black widor spider) & & & & & & & \\ _Limmorphisms hearts_ & & & & & & & \\ (caddisfy) & & & & & & & \\ _Oncyclous fscitats_ & & & & & & & \\ (large milkwheel bug) & GCA\_000696205.1 & Gras\_1.0 & 4047 & 339,960 & oncfas\_OGSv1.2 & 19,793 & 34.31\% & [34] \\ _Homalidasc viripennis_ & & & & & & & \\ (glassy-wired shapshooter) & & & & & & & \\ _Entermana diffins_ & & & & & & & \\ (calanoid copep) & & & & & & & \\ _Agrius planennis_ & & & & & & & \\ (emerald ash boorer) & & & & & & & \\ _Capitosoma foraminifera_ & & & & & & & \\ (parasatioid wasp) & & & & & & & \\ _Athalia roase_ & & & & & & & \\ (turning sawfly) & & & & & & & \\ _Certains omnita_ & & & & & & & \\ (Mediterranean fruit fly) & & & & & & & \\ _[PERSON]_ & & & & & & & \\ (Cimclade bed bug) & & & & & & & \\ _Virra destructor_ & & & & & & & \\ (parasitic mite) & & & & & & & \\ _Diaphrina citri_ & & & & & & & \\ (Asian citrus pyyllid) & NA & Version 3 & 749,525 & 40,596,296 & OGSv3 & 19,049 & 59.30\% & [43] \\ \hline \hline \end{tabular}
\end{table}
Table 3: Arthropod genomes selected for this study and their assembly and annotation statistics. The test species are sorted by the scaffold N50 value.  
Proteome sets for each species were downloaded from NCBI and functionally annotated using the workflow described above. For GOanna, we used the invertebrate reference databases, and only the GO terms with experimental evidence were assigned (-b). Custom BLAST parameters included a BLAST identity (-g) and query coverage (-q) cutoff of 70% with a maximum number of gap opening size (-k) of 9 to account for insertion or deletion of short peptides. Ideally, the query and BLAST match should be of identical length, but we allowed for some flexibility (-r 1.2) to account for natural diversity and potential assembly or annotation errors. InterProScan was run to identify InterPro domains, GO terms, and pathways for the input proteins (-g -l -p -c), and we used all the databases to extract the maximum amount of information possible. A single, comprehensive GAF was obtained by combining the results from GOanna and InterProScan. The same protein sets were then run through KOBAS [26] to annotate pathways. The KOBAS Annotate tool (-a) used the _D. melanogaster_ reference proteins (-s dme). The input data type must be specified (-t fasta:pro).