Supplemental materials for "Diploid and triploid chinook salmon (<em>Oncorhynchus tshawytscha</em>) have altered microRNA responses in immune tissues after infection with <em>Vibrio</em> <em>anguillarum</em> "

Supplemental files for study investigating the impact of <em>Vibrio</em> <em>anguillarum</em> infection on diploid and triploid Chinook salmon (<em>Oncorhynchus tshawytscha</em>) was investigated to identify if there was any significant immune regulation by microRNAs (miRNA). Small RNAs from hindgut (hg), head kidney (hk), and spleen (sp) were sequenced to determine if miRNA transcript abundance was altered due to ploidy and infection in nine-month old full-sibling diploids and triploids. MiARma-seq and bowtie1 were utilized to align reads to the Chinook salmon genome, and then read abundance associated with miRNA genes quantified using FeatureCounts. Read counts were then analyzed using DESeq2 to identify differentially expressed miRNA between ploidies as well as after infection in all three tissues. Finally, targets of differentially expressed miRNA were predicted using miRanda and pathway impacts predicted using KEGG analysis. <br> Files included: <strong>run_log.log</strong> - miARma-seq logfiles for hindgut (hg), head kidney (hk), and spleen (sp) <strong>stats.log</strong> - miARma-seq stats information for hindgut (hg), head kidney (hk), and spleen (sp) <strong>miarma.cadonic.known.tshawyt.sample.ini</strong> - initiation file for miARma-seq which includes all parameters that were utilized to analyze raw reads <strong>deseq2_cadonic_sample.R</strong> - sample of R code for DEseq2 used to identify differentially expressed miRNA genes <strong>Sasa_Ensembl_3UTR_clean.fasta </strong>- File of 3' untranslated region (UTR) for annotated genes in Ensembl biomart (Ensembl release 105) that was analyzed with miRanda (version v3.3a) to predict miRNA-mRNA relationships. Sequences with unavailable UTRs were cleaned by replacing empty spaces with N using "sed -e '/^[^&gt;]/s/[^ATGC]/N/g' Sasa_Ensembl_3UTR.txt &gt; Sasa_Ensembl_3UTR_clean.fasta".

本补充文件配套于一项旨在探究鳗弧菌（*Vibrio anguillarum*）感染对二倍体与三倍体奇努克鲑（*Oncorhynchus tshawytscha*）影响的研究，旨在明确微小RNA（microRNA，简称miRNA）是否介导了显著的免疫调控过程。研究人员针对9月龄全同胞二倍体与三倍体奇努克鲑的后肠（hg）、头肾（hk）及脾脏（sp）提取小RNA并开展测序，以解析miRNA转录本丰度是否因倍性与感染状态发生改变。
实验分析流程如下：本研究采用miARma-seq与bowtie1工具将测序reads比对至奇努克鲑参考基因组，随后通过FeatureCounts定量miRNA基因对应的reads计数；继而使用DESeq2对reads计数数据进行差异表达分析，以鉴定不同倍体组间以及感染后三种组织中差异表达的miRNA。此外，本研究通过miRanda预测差异表达miRNA的靶基因，并借助KEGG分析预测相关调控通路的影响。
本次补充文件包含以下内容：
- **run_log.log**：后肠（hg）、头肾（hk）及脾脏（sp）的miARma-seq运行日志文件
- **stats.log**：后肠（hg）、头肾（hk）及脾脏（sp）的miARma-seq统计信息文件
- **miarma.cadonic.known.tshawyt.sample.ini**：miARma-seq初始化配置文件，包含分析原始reads所使用的全部参数
- **deseq2_cadonic_sample.R**：用于鉴定差异表达miRNA基因的DESeq2示例R代码
- **Sasa_Ensembl_3UTR_clean.fasta**：针对Ensembl生物Mart（Ensembl版本105）中注释基因的3'非翻译区（3' untranslated region，UTR）构建的序列文件，该文件用于miRanda（v3.3a版本）预测miRNA与mRNA的调控关系。对于无法获取UTR序列的位点，通过命令`sed -e '/^[^>]/s/[^ATGC]/N/g' Sasa_Ensembl_3UTR.txt > Sasa_Ensembl_3UTR_clean.fasta`将非ATGC的空白字符替换为N以完成序列清洗。