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1) IsomiR Identification

Plant IsomiR Atlas (PIA) is a database depositing isomiRs identified from plant landscape. For this version v1.0, PIA deposits 196,829 unique isomiR signatures (98,734 unique isomiR sequences) identified from 6,167 plant miRNA hairpins by using 667 Illumina small RNA sequencing datasets of 23 species, whose genomes, primary transcripts and annotation information are mostly from phytozome, except those of Nelumbo nucifera, which are from lotus-db (Table 1).

Table 1 Genome backgrounds we used for isomiR identification

Species	Source	Genome	Transcript	GO Background
Amborella trichopoda	PhytozomeV11	Atrichopoda_291_v1.0.fa.gz	Atrichopoda_291_v1.0.transcript_primaryTranscriptOnly.fa.gz	Atrichopoda_291_v1.0.annotation_info.txt
Arabidopsis lyrata	PhytozomeV11	Alyrata_107_v1.fa.gz	Alyrata_107_v1.0.transcript_primaryTranscriptOnly.fa.gz	Alyrata_107_v1.0.annotation_info.txt
Arabidopsis thaliana	PhytozomeV11	Athaliana_167_TAIR9.fa.gz	Athaliana_167_TAIR10.transcript_primaryTranscriptOnly.fa.gz	Athaliana_167_TAIR10.annotation_info.txt
Brachypodium distachyon	PhytozomeV10	Bdistachyon_283_assembly_v2.0.fa.gz	Bdistachyon_283_v2.1.transcript_primaryTranscriptOnly.fa.gz	Bdistachyon_283_v2.1.annotation_info.txt
Brassica rapa	PhytozomeV11	BrapaFPsc_277_v1.fa.gz	BrapaFPsc_277_v1.3.transcript_primaryTranscriptOnly.fa.gz	BrapaFPsc_277_v1.3.annotation_info.txt
Carica papaya	PhytozomeV11	Cpapaya_113_r.Dec2008.fa.gz	Cpapaya_113_ASGPBv0.4.transcript_primaryTranscriptOnly.fa.gz	Cpapaya_113_ASGPBv0.4.annotation_info.txt
Citrus clementina	PhytozomeV11	Cclementina_182_v1.fa.gz	Cclementina_182_v1.0.transcript_primaryTranscriptOnly.fa.gz	Cclementina_182_v1.0.annotation_info.txt
Citrus sinensis	PhytozomeV11	Csinensis_154_v1.fa.gz	Csinensis_154_v1.1.transcript_primaryTranscriptOnly.fa.gz	Csinensis_154_v1.1.annotation_info.txt
Glycine max	PhytozomeV11	Gmax_275_v2.0.fa.gz	Gmax_275_Wm82.a2.v1.transcript_primaryTranscriptOnly.fa.gz	Gmax_275_Wm82.a2.v1.annotation_info.txt
Gossypium raimondii	PhytozomeV11	Graimondii_221_v2.0.fa.gz	Graimondii_221_v2.1.transcript_primaryTranscriptOnly.fa.gz	Graimondii_221_v2.1.annotation_info.txt
Malus domestica	PhytozomeV11	Mdomestica_196_v1.0.fa.gz	Mdomestica_196_v1.0.transcript_primaryTranscriptOnly.fa.gz	Mdomestica_196_v1.0.annotation_info.txt
Manihot esculenta	PhytozomeV11	Mesculenta_305_v6.fa.gz	Mesculenta_305_v6.1.transcript_primaryTranscriptOnly.fa.gz	Mesculenta_305_v6.1.annotation_info.txt
Medicago truncatula	PhytozomeV11	Mtruncatula_285_Mt4.0.fa.gz	Mtruncatula_285_Mt4.0v1.transcript_primaryTranscriptOnly.fa.gz	Mtruncatula_285_Mt4.0v1.annotation_info.txt
Nelumbo nucifera	http://lotus-db.wbgcas.cn/	Lotus_mega_gap1M.fa	lotus_marker_all_mega.gff.CDS	gene.go.tbl
Oryza sativa	PhytozomeV11	Osativa_323_v7.0.fa.gz	Osativa_323_v7.0.transcript_primaryTranscriptOnly.fa.gz	Osativa_323_v7.0.annotation_info.txt
Populus trichocarpa	PhytozomeV11	Ptrichocarpa_210_v3.0.fa.gz	Ptrichocarpa_210_v3.0.transcript_primaryTranscriptOnly.fa.gz	Ptrichocarpa_210_v3.0.annotation_info.txt
Setaria italica	PhytozomeV11	Sitalica_312_v2.fa.gz	Sitalica_312_v2.2.transcript_primaryTranscriptOnly.fa.gz	Sitalica_312_v2.2.annotation_info.txt
Solanum lycopersicum	PhytozomeV11	Slycopersicum_225_iTAGv2.40.fa.gz	Slycopersicum_225_iTAGv2.3.transcript_primaryTranscriptOnly.fa.gz	Slycopersicum_225_iTAGv2.3.annotation_info.txt
Solanum tuberosum	PhytozomeV11	Stuberosum_206_v3.fa.gz	Stuberosum_206_v3.4.transcript_primaryTranscriptOnly.fa.gz	Stuberosum_206_v3.4.annotation_info.txt
Sorghum bicolor	PhytozomeV11	Sbicolor_313_v3.0.fa.gz	Sbicolor_313_v3.1.transcript_primaryTranscriptOnly.fa.gz	Sbicolor_313_v3.1.annotation_info.txt
Triticum aestivum	PhytozomeV11	Taestivum_296_v2.fa.gz	Taestivum_296_v2.2.transcript_primaryTranscriptOnly.fa.gz	Taestivum_296_v2.2.annotation_info.txt
Vitis vinifera	PhytozomeV11	Vvinifera_145_Genoscope.12X.fa.gz	Vvinifera_145_Genoscope.12X.transcript_primaryTranscriptOnly.fa.gz	Vvinifera_145_Genoscope.12X.annotation_info.txt
Zea mays	PhytozomeV11	Zmays_284_AGPv3.fa.gz	Zmays_284_5b+.transcript_primaryTranscriptOnly.fa.gz	Zmays_284_5b+.annotation_info.txt

The species-specific hairpin and mature sequences used for isomiR identification are from miRBase and Plant Non-coding RNA Database. We integrated miRNAs sequences in these two databases and removed the redundancy. Most datasets are from the NCBI SRA database and transformed into collapsed FASTA files using in-house Perl script which calls cutadapt to remove adapter sequences accurately. Clean reads are then used for isomiRs identification by a Perl script of modified isomiR2Function called isomiRIden. Briefly, sequenced reads and canonical miRNAs are mapped on species-specific pre-miRNAs allowing no mismatch. By comparing mapping information, templated isomiRs as well as their relative position to canonical miRNAs are identified. After that, reads not mapped on precursors are mapped on genome allowing no mismatch. Reads not mapped on the genome are then been mapped on species-specific pre-miRNAs again allowing two mismatches. By comparing mapping information, non-templated isomiRs as well as their relative position to canonical miRNAs and mismatch positions are identified. Finally, by analyzing the identified information, isomiRs are indexed and accurately classified into different categories (Figure 1). You can read our paper " isomiR2Function: An Integrated Workflow for Identifying MicroRNA Variants in Plants" for more information.

2) Structure of Plant IsomiR Atlas

PIA was implemented in MySQL, PHP, JavaScript and Perl. Anyone can access this database totally free. The MySQL database of PIA consists of seven tables which are seq, hairpin, exist, evidence, isomiR_alignment, mature_alignment, precursor_alignment, targetfinder, psRNATarget, miRNA_targetfinder and datasets. database is an independent table (Figure 2) which stores the information of datasets. Relation between other tables and their column information are shown in the following Figure 3, where 'P' is for primary key, 'I' is for indexed and 'F' is for foreign key. The same name columns in different columns with a 'F' reference to same name column without 'F'. For example, seq.ID is the foreign key of tables evidence.ID, hairpin.ID, targetfinder.ID and psRNATarget.ID.