Journal of Biotechnology and Sericology 89, 1-7 (2020)

Development of an application, Bombyx mori tool for ortholog picking (BmTOP)

Mizuki Ohno1, Munetaka Kawamoto2 and Ken Sahara1, 3＊

1 Division of Biocontrol and Bioenvironmental Sciences, United Graduate School of Agricultural Sciences, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan 2 Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan 3 Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka 020-8550, Japan (Received September 6, 2019; Accepted October 24, 2019)

We have developed an application to select Bombyx mori orthologs from assembled RNA sequencing (RNA- seq) data in species of interest. The application, BmTOP (Bombyx mori tool for ortholog picking) is composed of 6 steps, 1) tblastx analysis using B. mori gene models 2017, 2) alignment of the first hit and assignment of the B. mori gene data, 3) elimination of multi-copy genes in species of interest, 4) elimination of data by less than a score and/or more than an expect value (e-value), 5) elimination of data when value of first hit divided by second hit is not less than a threshold, and 6) comparison with B. mori single-copy gene list to exclude B. mori multi-copy genes. As default, e-value not more than 1e−10 is set at Step 1, score 100 and e-value 1e−80 at Step 4, and result of calculation 1e−80 at Step 5. We subjected the newly read erate RNA-seq data that carry 64,764 contigs with an N50 of 1,357 bp to BmTOP analysis. BmTOP selected 1,978 C. erate contigs as B. mori single-copy gene orthologs. The in-silico mapping of the orthologs covers all 28 B. mori chromosomes. The re- sults are promising for successful primer design to select bacterial artificial chromosomes containing orthologs for gene-based comparative mapping using fluorescence in situ hybridization. Key words: bioinformatics, Eastern pale clouded yellow (Colias erate), orthology inference, RNA-seq, silk- worm (Bombyx mori)

2012) requires more sophisticated and innovative tools to INTRODUCTION process RNA-seq data. , an insect order including moths and but- For FISH mapping on Lepidoptera, genomic libraries terflies, has holokinetic chromosomes (Melters et al., (i.e., BAC or fosmid library) have been constructed to se- 2012). Comparative gene mapping and genome sequencing lect DNA probes (Yasukochi et al., 2009; 2016; Yoshido have helped uncover chromosomal co-linearity and con- et al., 2011; Sahara et al., 2013). For probe selection, one served gene order in Lepidoptera (Pringle et al., 2007; needs sequence-tagged site primer pairs of orthologous van’t Hof et al., 2008; 2013; Yasukochi et al., 2009; 2016; genes (Kamimura et al., 2012) from B. mori. Single-copy Baxter et al., 2011; Yoshido et al., 2011; Sahara et al., gene orthologs are most preferred for comparative map- 2013; Ahola et al., 2014). While recent studies showed ping. Hence, developing a tool to select B. mori single- that unprecedented reorganization had occurred in white copy genes from RNA-seq data of non-model lepidopteran (Nallu et al., 2018; Hill et al., 2019). To under- has been urgently required for FISH mapping in stand chromosome evolution in Lepidoptera comparative moths and butterflies. genome studies in wider species are inevitable. Since We developed a new tool to efficiently identify B. mori comparative physical (e.g., bacterial artificial chromo- single-copy gene orthologs, which are particularly useful somes-fluorescence in situ hybridization (BAC-FISH)) and for BAC or fosmid screening, from RNA-seq data. The recombination based (e.g., molecular linkage analysis) tool feasibly identifies B. mori single-copy gene orthologs mapping requires orthologous gene information, genome from Colias erate RNA-seq data. data from the model Lepidoptera, Bombyx mori (Mita et al., 2004; Xia et al., 2004; International Silkworm Ge- MATERIALS AND METHODS nome Consortium, 2008; Kawamoto et al., 2019), have contributed greatly to chromosome evolution research in Insects Lepidoptera. Moreover, since RNA sequencing (RNA-seq) We collected adult Colias erate females from a field in using next generation sequencing (NGS) technology has Iwate University, reared them, and collected eggs that replaced Sanger sequencing of cDNA library, selecting B. were laid in our laboratory. The hatched progenies were mori orthologs from non-model species (Kamimura et al., fed on red (Trifolium pratense) and white (T. re-  pens). Day 4 pupae were stored in RNA Later (Thermo *To whom correspondence should be addressed. Fisher Scientific, Waltham, MA, USA) at −20°C to be Fax & Tel: +81-19-621-6147. used later for RNA extraction. Email: [email protected] 2 Ohno et al.

Fig. 1. Six step process in BmTOP analysis. Text in rectangle and rounded-rectangle boxes describes the flow and treatment, respectively.

Transcriptome data search against the B. mori gene models 2017 (Kawamoto Total RNA was extracted from a male and a female us- et al., 2019, http://silkbase.ab.a.u-tokyo.ac.jp) creates a “hit ing Nucleospin® RNA (Macherey-Nagel, Düren, Germany). table” from assembled transcriptome data, which have ho- Hokkaido System Science Co. Ltd. carried out stranded mology with expect value (e-value) not more than 1e−10 mRNA-seq by Hiseq 2500 (Illumina, 1 lane, PE-101 bp, (Threshold 1, default), 2) Processing: this step adds B. 4 Gb). Raw read data were preprocessed using Cutadapt mori information to the hit table and top hits from each (https://cutadapt.readthedocs.io/en/stable/) and Trimmo- BLAST result are then extracted and listed, 3) Removal matic (http://www.usadellab.org/cms/?page=trimmomatic), of multi-copy genes in species of interest: multiple con- and assembled by running Trinity (Grabherr et al., 2011). tigs showing homology to the same B. mori gene ID are excluded because they are unlikely to be single-copy gene Evaluation of B. mori single-copy gene orthologs orthologs. With this step, contigs containing multiple ho- identified by BmTOP mologs can be eliminated, 4) Selection of genes with high To confirm whether BmTOP was able to identify ortho- homology: if e-value and/or score of top hits do not fulfill logs fitting the requests, B. mori single-copy gene ortho- the criteria (Threshold 2: score, not less than 100; e-value, logs of C. erate were selected by SonicParanoid (Cosentino not more than 1e−80), BmTOP discards the data, 5) Elimi- and Iwasaki 2018, http://iwasakilab.bs.s.u-tokyo.ac.jp/ nation of all possible multiple homologs: if the condition sonicparanoid/) (Run mode: fast), using deduced amino “top hit e-value / second hit e-value” is not less than 1e−80 acid data from C. erate RNA-seq data using EMBOSS (Threshold 3)”, BmTOP discards the hit data. BmTOP getorf (http://emboss.sourceforge.net/apps/cvs/emboss/ also discards the hit data when the e-value of second hit apps/getorf.html). We calculated the share frequency of is less than 1e−50 and of top hit is 0.0, and 6) Elimination the data identified by BmTOP with the orthologs used in of possible B. mori multi-copy genes: to avoid picking B. Misof et al. (2014). mori multi-copy genes, we carried out the first five steps using BmTOP with B. mori gene models 2017 as query. If B. mori orthologs from the top hit data do not appear in RESULTS the results, they are excluded. All thresholds can be Development of BmTOP changed individually from the default. BmTOP can pro- An application called Bombyx mori tool for ortholog cess a combination of 16 conditions at once, for each 4 e- picking (BmTOP, https://github.com/mzkohno/BmTOP) value setting of thresholds 2 and 3. selects single-copy B. mori gene orthologs from an as- sembled transcriptome data. BmTOP composes of 6 steps Selection of the single-copy B. mori gene ortho- as shown in Fig. 1; 1) Homology search by BLAST anal- logs from the C. erate transcriptome data ysis using NCBI BLAST+ v2.2.31 tblastx search: The We obtained 4,053 Mb Colias erate RNA-seq data. Bombyx mori tool for ortholog picking 3

Fig. 2. The histogram shows the number of Bombyx mori genes mapped in silico for which the Colias erate ortho- logs were detected using BmTOP analysis with the default setting.

Trinity generated 64,764 de novo assembled contigs with thologous genes between B. mori and C. erate with an N50 of 1,357 bp. We used the contigs as a query for SonicParanoid default settings. Using the most sensitive BmTOP analysis. During the analysis, the first 2 steps de- analysis (Table 1), 1,790 out of 1,978 (90.5%) BmTOP tected 17,448 top hit contigs as candidates and 13,624, selected orthologs were shared with the orthologs of 1,527, 206 and 113 candidates were eliminated through SonicParanoid selected (Fig. 4A). The share frequency the 3rd to 6th steps respectively. The analysis extracted was similar when the most tolerant analysis applied 1,978 candidate contigs carrying single-copy B. mori gene (89.6%, 2,453 out of 2,738). orthologs which have from 18 to 129 orthologs on each B. Misof et al. (2014) constructed a phylogenetic tree of mori chromosome (Fig. 2). The chromosomal distribution insects using widely conserved gene orthologs from ar- was shown by counts at every 500 kb of B. mori chromo- thropods. The share frequency for genes selected by somes (Fig. 3). We also analyzed 15 threshold conditions BmTOP analysis was 24.8% (490 out of 1,978) (Fig. 4B) in addition to the default condition (e-value combinations and 20.5% (561 out of 2,738) in most sensitive and toler- of 1e−20, 1e−40, 1e−60, and 1e−80 in thresholds 2 and 3). ant analyses respectively. BmTOP identified more candidates with less stringent combination of thresholds selected (Table 1). DISCUSSION Distribution of the identified candidates Comparative chromosomal analysis with the help of To visualize the positions of the B. mori genes, we de- BAC-FISH mapping in Lepidoptera requires comprehen- scribed a script, gene_plotter.R (https://github.com/nkimoto/ sive single-copy B. mori gene ortholog coverage on all gene_plotter/) based on Bioconductor karyoploteR (http:// chromosomes (Yasukochi et al., 2009). The genome size bioconductor.org/packages/release/bioc/html/karyoploteR. of the Phoebis sennae, which belongs to the same sub- html). Using this script, the genes listed by BmTOP are family as Colias erate, Coliadinae, is about 345 Mb (Cong placed on putative B. mori chromosomes (Length_Silkbase. et al., 2016). The genome size of Pieris rapae is estimat- Sheet1.tsv). The selected genes, which are orthologs of C. ed to be 246 Mb (Shen et al., 2016) and 272 Mb (Nallu erate genes identified by BmTOP, were located within et al., 2018). Hill et al. (2019) reported the genome size of 2,414,428 bp from the chromosomal end (the longest dis- P. napi to be 299 Mb. The genome size of pierid species tance with no genes was from KWMTBOMO09323 to the is about 53-75% compared to that of B. mori (460.3 Mb) end of Chr15, Fig. 3, gray arrow). The largest distance with (Kawamoto et al., 2019). The B. mori orthologs of C. er- no genes in a chromosome was 2,290,030 bp and was pres- ate genes selected in this study showed full-coverage for ent between KWMTBOMO03496 and KWMTBOMO03557 B. mori chromosomes (Fig. 3) and their numbers were in Chr6 (Fig. 3, black arrow). sufficient for gross genome comparison using FISH map- ping in C. erate (n = 31) if the genome size is similar to Comparison of the identified dataset those species in . In this regard, BmTOP works SonicParanoid is an analysis tool for ortholog detection well for our purpose. (Cosentino and Iwasaki, 2018). We detected 10,121 or- The number of orthologs detected with BmTOP was 4 Ohno et al.

Fig. 3. Chromosomal distribution of Bombyx mori genes mapped in silico for which the Colias erate orthologs were identified using BmTOP analysis with the default setting. The number of genes shown was counted at every 500 kb window. Bars below each graph represent the B. mori chromosome length (Mbp). Black arrow indicates the region of the largest distance with no gene between two genes in a chromosome. Gray arrow indicates the region of longest distance with no genes from a chromosome end. only 19.5% (1,978 / 10,121) of that with SonicParanoid higher homologous orthologs between C. erate and B. (Fig. 4A) because the third step in BmTOP critically elim- mori. The feature provided that approximate 24.8% (490 / inated possible multiple homologs (13,624 out of 17,448 1,978) genes selected by BmTOP also appeared in the or- candidates). Because our need is to pick not larger num- thologs listed in Misof et al. (2014) (Fig. 4B). Although ber but more accurate single gene orthologs, the unique several ortholog analysis applications have been published step in the pipeline is fits our purpose. Together with the (Ebersberger et al., 2009; Emms and Kelly 2015; Cosentino subsequent stringent elimination steps, BmTOP detected and Iwasaki 2018), these features are considered to be Bombyx mori tool for ortholog picking 5

Table 1. Number of Colias erate genes detected using the BmTOP analysis with dif- ferent combination of thresholds 2 and 3. Threshold 1 was kept at an e-value of 1e−10

threshold 2 −20 −40 −60 −80 1e 1e 1e 1e threshold 3 1e−20 2,738 2,612 2,362 2,032 1e−40 2,700 2,575 2,336 2,018 1e−60 2,666 2,541 2,306 2,001 1e−80 2,642 2,517 2,282 1,978

Fig. 4. Venn diagram comparison between orthologous gene numbers selected by BmTOP and by SonicParanoid (A). Venn diagram comparison between orthologous gene numbers selected by BmTOP and number of or- thologs described in Misof et al. (2014) (B). unique to BmTOP. been published. However, if query data are small, detec- The number of orthologs selected by BmTOP did not tion of orthologous genes cannot be guaranteed owing to always depend on the chromosome length and/or gene the characteristics of this application (Step 3). To get ac- number (Figs. 2, 3). For example, B. mori Chr15 and Chr22 curate results, data applied to BmTOP should have large were of similar lengths, 18,440,292 bp and 18,482,526 bp genome-wide coverage. respectively. The predicted gene numbers were 804 and We selected single-copy gene orthologs for BAC screen- 677 for Chr15 and Chr22, respectively. The number of se- ing using C. erate RNA-seq data. Orthologs containing lected orthologs was 124 and 70 for Chr15 for Chr22 by unique sequences are required for BAC screening because BmTOP, while 535 and 376 by SonicParanoid, respective- common motifs and/or repeats sometimes result in multi- ly. These results reflect that single-copy genes are not- al ple signals by BAC-FISH. BmTOP is suitable for this ways equally distributed, especially highly homologous BAC-FISH prerequisite. Although several applications for single-copy genes. orthology analysis have been developed (Ebersberger et BmTOP selects B. mori orthologs that do not share al., 2009; Emms and Kelly, 2015; Cosentino and Iwasaki, similar domains because it is designed to remove contigs 2018), an application developed according to requirement containing high homology sequences in a genome and/or is effective in selecting information from big data. The transcriptome data. This method reduces the possibility of single-copy B. mori gene orthologs are used not only for detecting false orthologs in the screening procedure and chromosome comparison studies in Lepidoptera but also in hence, cannot detect genes with similar motifs and/or re- Trichoptera (Fujimoto et al., 2018). An application called peat sequences. For example, although Hox genes are BmTOP, can identify single-copy B. mori gene orthologs widely conserved as single-copy genes in all , to support comparative genome researches. BmTOP can except in scorpions such as Mesobuthus martensii (Di et be applied to other models if the reference gene model is al., 2015), they cannot be detected by BmTOP because replaced by the model of interest. each gene has a 180 bp homeobox motif. The number of orthologs detected on B. mori Chr6 that carries the Hox ACKNOWLEDGMENTS cluster, is fewer than other B. mori chromosomes that have similar length and gene number (Figs. 2, 3). We thank N. Kimoto for valuable advice to describe the BmTOP is designed to process not only assembled script, gene_plotter.R. A part of this research was support- RNA-seq data, but also gene models, cDNAs, and expres- ed by JSPS (Kakenhi: 16H05050 and 19K06067). sion sequence tag (EST) sequences. Thus, it is possible to use data from species whose sequence information has 6 Ohno et al.

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