TITLE: Methods in &#34;The first released available genome of the common ice plant (Mesembryanthemum crystallinum L.) extended the research region on salt tolerance, C3-CAM photosynthetic conversion, and halophism&#34;
AUTHORS: Ryoma Sato, Yuri Kondo, Sakae Agarie

[DESCRIPTION]
    The wild-type seeds of the common ice plant were sowed on a germination medium. The seedlings were grown in particular soil and treated with a solution that included salt and nutrients for two weeks in a greenhouse. Genomic DNA was extracted from frozen leaves and made into a library using special kits. Obtained NGS data were trimmed and assembled by Musket, ALGA, and Redundans. The completeness of the genome was checked using BUSCO and BLASTN.                
    In this protocol, five types of analysis methods were introduced, including the establishment of a phylogenetic tree based on 18S rDNA via NGPhylogeny.fr, detection of repetitive regions with RepeatModeler2, TEclass, and RepeatMasker, search for genomic sequences coding tRNA and miRNA by tRNAscan-SE2.0 and Infernal, gene prediction using BRAKER2 and DIAMOND, and protein domain searches based on Pfam database using HMMER. 

[STEPS]
SECTION: Table of contents
1. ① DNA extraction, library construction, and sequencing
② Clean read preparation and genome size estimation
➂ De novo genome assembly and quality evaluation
④ Phylogenetic tree creation among multiple plant species using 18S ribosomal DNA sequences
⑤ Detection of repetitive regions
➅ Search for genomic sequences coding transfer RNA (tRNA) and micro-RNA (miRNA)
⑦ Gene prediction
⑧ Protein domain searches
SECTION: ① DNA extraction, library construction, and sequencing
2.     Total genomic DNA was extracted from the leaf tissue and purified using MagExtractor™-Plant Genome Nucleic Acid Purification Kits (Toyobo Co., Ltd., Shiga, Japan), according to the manufacturer’s instructions. The DNA samples were fragmented by sonication and used to construct short insert paired-end libraries construction using NEBNext® Ultra™DNA Library Prep Kits for Illumina (New England Biolabs Ltd., Ipswich, MA, USA). Briefly, in the end-repair step, fragmented DNA was phosphorylated at the 5' end and adenylated at the 3' end. During the ligation step, full-length circulated adaptor sequences were ligated to the fragments. After adaptor cleavage, purification, and size selection were performed. The indexed PCR products were taken to obtain the final sequencing libraries. The mean insert size for paired-end libraries was 300 bp. The paired-end (2×150 bp) sequencing was conducted on an Illumina NovaSeq 6000 platform (Illumina Inc., San Diego, CA, USA). 
SECTION: ② Clean read preparation and genome size estimation
3.     The mean insert size was calculated using REAPR (v1.0.18)(Hunt et al. 2013), and raw paired-end sequences were filtered based on the frequency of 21-mer sequences using the program Musket (v1.1)(Liu et al. 2013). 
The key parameter values were as follows: musket -omulti output -inorder pair1.fastq pair2.fastq. 
 
    
    Sequence reads that appeared rarely or abnormally frequently were removed to obtain clean read data. In the corrected reads, unique and duplicate read numbers in the corrected reads were measured using fastqc (v0.11.9)(Simon 2010). The clean data were used for an estimate of genome size as follows. 

 

    K-mers were counted and exported to histogram files using jellyfish (v2.3)(Marçais and  Kingsford 2011) [key parameter: jellyfish histo reads.jf]. 
GenomeScope2.0(Ranallo-Benavidez et al. 2020) corresponding key parameters were applied to calculate the genome sizes using k-mers lengths of 21 and 25.

 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              
SECTION: ➂ De novo genome assembly and quality evaluation
4.     The reads were assembled using ALGA (v1.0.3; Swat et al. 2021) with the default parameter --error-rate = 0.02. long DNA fragments 1 to 10 kb in length were combined, and gaps between them were filled with unknown bases (Ns) using Redundant (v0.14a;Pryszcz and  Gabaldón 2016), a software program for scaffolding, with default parameter values. 

 

    The genome coverage of reads was estimated using the Mosdepth program (Pedersen and Quinlan 2018). 

 

    The completeness of the assembled genome was evaluated based on the content of orthologs in higher plants, using the benchmarking universal single-copy orthologs (BUSCO) program (v5.0; Manni et al. 2021). The lineage dataset was embryophyta_odb10 (creation date: 2020-09-10, number of BUSCOs: 1614). 

 

    We also searched for core genes in the genome sequences of nine other plant species: Kewa caespitosa, Pharnaceum exiguum, Macarthuria australis, Solanum chaucha, Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa using BUSCO. The first three species belong to the same order, Caryophyllales, to which the ice plants belong. Genome information was obtained from the NCBI (see Supplementary Note 1 “Address to genome information”;  Supplementary_Information: Sato et al., 2022a). The number of bases, sequences, sequences in several base number ranges, and the maximum base length of the final draft genome sequences was calculated using gVolante (v2.0.0)(Nishimura et al. 2017). BLASTN (v2.2.31+; McGinnis and  Madden 2004) was used to investigate the number of cDNA sequences identified by transcriptome (Lim et al. 2019), and registered DNA sequences (retrieved from NCBI, last accessed February 2022) were aligned to the final assembled genome sequence.                                                                                                                                                                                                                                                                                                                                                             
SECTION: ④ Phylogenetic tree creation among multiple plant species using 18S ribosomal DNA sequences
5.     The 18S ribosomal genes were extracted using barrnap (v0.9; Seemann 2018) from the obtained genome sequences of the ice plant. 
 
    As comparative objectives, 25 kinds of 18S ribosomal genes from general crops (Japanese radish [Raphanus sativus], Soybean [Glycine max], Japanese trefoil [Lotus japonicus], Barrelclover [Medicago truncatula], Adzuki bean [Vigna angularis], Banana [Musa acuminata], Barley [Hordeum vulgare], Sorghum [Sorghum bicolor], Bread wheat [Triticum aestivum], Maize [Zea mays], Apple [Malus domestica], Peach [Prunus persica], Coffee tree (Arabica var.) [Coffea arabica], Coffee tree (Robusta var.)[C. canephora], Clementine [Citrus clementina], Orange [C. sinensis], Poplar, Tobacco [Nicotiana tabacum], Tomato [Solanum lycopersicum], Eggplant [S. melongena], Potato [S. tuberosum] and Grape [Vitis vinifera]) were selected using the SILVA database (Release. 2020-08; Pruesse et al. 2007). After joining all ribosomal DNA sequences into one file, a molecular phylogenetic tree was created using implemented in NGPhylogeny.fr (Lemoine et al. 2019) (Released in 2019). SH-aLRT (Shimodaira-Hasegawa-approximate likelihood ratio test) (Shimodaira and Hasegawa 1999) was used to determine the molecular phylogenetic tree.                                                         
SECTION: ⑤ Detection of repetitive regions
6.     Repetitive sequences were detected, and custom repeat libraries involving transposable elements and long terminal repeat-retro transposons were generated using RepeatModeler2 (v2.0.2; Flynn et al. 2020) and TEclass (v2.1.3)(Abrusán et al. 2009). Known repeat sequences were detected and classified in the assembled genome sequence with reference to the Repbase library(Bao et al. 2015) and the custom repeat libraries, using RepeatMasker(v4.1.2-p1; Smit et al. 2013-2015). The capital letters in the genome sequences were replaced with small characters as soft masking.

 
                                                                                                                                                   
SECTION: ➅ Search for genomic sequences coding transfer RNA (tRNA) and micro-RNA (miRNA)
7.     The tRNA genes were identified in the draft common ice plant genome using tRNAscan-SE2.0 (v2.0.9)(Chan et al. 2021). 

 

    The tRNA data of other nine plant species—Arabidopsis, rice, tomato, poplar, horseradish, potato, grape, soybean, and coffee tree (robusta species)—were obtained from the PlantRNA database (Cognat et al. 2013). The percentages of arbitrary tRNAs against the total tRNAs in the genome were calculated and compared to the ice plants' values with those of the other species. Smirnov-Grubbs' outlier tests were performed to select tRNAs more significantly involved. The test statistic T was calculated using the following equation:

  

    The miRNA loci in the genome sequence were identified using the cmscan command in infernal (v1.1.4; Nawrocki and  Eddy 2013) using Rfam.

 

SECTION: ⑦ Gene prediction
8.     The BRAKER2 pipeline (v2.1.5; Brůna et al. 2021) was used for the prediction of genes in the common ice plant genome. Amino acid sequences were translated from the transcriptome profile reported by Lim et al. (2019) and used as additional reference data for the prediction of genes. BRAKER2 was used with the default parameters (–softmasking). 

 

    The total sequences, total bases, total amino acids, and N50 were computed based on the resulting fasta-format files containing information about the genes, coding sequences, and amino acids using seqkit (v2.0.0; Shen et al. 2016) [key parameter: seqkit stats]. Protein BLAST searches (E-value < 1e-5) were conducted using DIAMOND (v2.0.13.151; Buchfink et al. 2021) against the NCBI-non-redundant protein sequences (retrieved from NCBI in March 2022), Uniprot-swissprot(retrieved in March 18), Ensemble TAIR10 (retrieved in March 2022), and NCBI poplar amino acid sequence databases(retrieved from NCBI in March 2022).

 

SECTION: ⑧ Protein domain searches
9.     The protein domains in the genome were identified using the Pfam (v33.1) database(Mistry et al. 2021) with E-value < 1e-3, using HMMER (v3.1b2; Potter et al. 2018). 

 

    The protein databases of rice, maize, and poplar from the NCBI (last accessed February 2022) were used in the domain for a detailed classification of the PKinase family, the iTAK (v18.12) web tool (Zheng et al. 2016; last accessed February 2022) was utilized. The ratio of families with a high ratio of genes to total genes in the ice plant was compared with that of the same families in the other plants. For statistical analysis, we used Smirnov-Grubbs' outlier tests. The following equation was used to obtain the test statistic T:

    


    Finally, BLASTP was used to compare proteins generated from the ice plant genome and those from Arabidopsis, rice, maize, and poplar and renamed TAIR10 ID. These IDs were subjected to gene ontology (GO) enrichment analysis using DAVID (updated in 2022; accessed on March 24; Sherman et al. 2022) based on a modified Fisher exact probability test with E-value < 0.05.