Deep Sequencing-Based Transcriptome Analysis of Chicken Spleen in Response to J Subgroup Avian Leukosis Virus (ALV-J) Infection.

Purpose: The goals of this study are to investigate the differentially expressed genes between ALV-J infected (WRR+) and uninfected (WRR-)chickens spleens by Illumina deep sequencing. Methods: 140-day-old female chickens of White Recessive Rock (WRR) were confirmed as J subgroup avian leukosis virus (ALV-J) infection. Total RNA from three ALV-J-infected spleens (designated: WRR1+, WRR2+, WRR3+) and three uninfected normal spleen samples (designated: WRR1-, WRR2-, WRR3-) was isolated by TRIzol following the manufacturer’s instruction (Invitrogen, CA, USA). RNA samples of three individuals within each group were pooled with equal amounts, and then were subjected to Illumina deep sequencing by Illumina Genome Analyzer IIx. Results: Through raw data processed, 49,979,648 and 43,704,401 clean reads with an average length of 101 bp, which represented total residues of 4,859,084,087 and 4,238,826,168 bp, were obtained for WRR- and WRR+ libraries, respectively. Subsequently, the clean reads in the two libraries were assembled. Altogether, 121,493 contigs were assembled with an average length of 927 bp (ranged from 300 bp to 23,402 bp), leading to generation of 82,829 unigenes. The length of unigenes varied from 351 bp to 28,928 bp, with an average length of 1,155 bp. Based on the FPKM value of each gene, 252 DEGs were identified by DEGseq package using Benjamini-q-value of 0.05 as a cut-off. In ALV-J infected spleens, 90 genes showed up-regulated and 162 showed down-regulated expression when compared to uninfected samples. Conclusions: Our study represents the first time to elucidate the ALV-J infected chickens’spleens at the transcription level by RNA-seq technology. A total of 252 genes were found to be differentially expressed in ALV-J infected spleens when compared to uninfected chickens. These genes can be considered as candidates for further study ALV-J invasion. Spleen mRNA profiles of 140-day-old ALV-J infected (WRR+) and uninfected (WRR-) female chickens of White Recessive Rock were generated by deep sequencing, using Illumina Genome Analyzer IIx.

研究目的：本研究旨在通过Illumina深度测序，探究J亚群禽白血病病毒（ALV-J）感染组（WRR+）与未感染组（WRR-）鸡脾脏组织间的差异表达基因。
研究方法：选取140日龄的隐性白洛克鸡（White Recessive Rock，简称WRR），确认其感染J亚群禽白血病病毒（ALV-J）。按照生产商操作说明，采用TRIzol试剂（Invitrogen，美国加利福尼亚州）分别提取3份ALV-J感染脾脏样本（编号：WRR1+、WRR2+、WRR3+）与3份未感染正常脾脏样本（编号：WRR1-、WRR2-、WRR3-）的总RNA。将每组3个个体的RNA样本按等量混合后，利用Illumina Genome Analyzer IIx测序平台开展Illumina深度测序。
研究结果：对原始数据进行质控过滤后，WRR-文库与WRR+文库分别获得49,979,648条和43,704,401条clean reads，平均读长为101 bp，总碱基量分别为4,859,084,087 bp与4,238,826,168 bp。随后对两个文库的读段进行拼接组装，共获得121,493条重叠群（contigs），平均长度为927 bp（范围300 bp至23,402 bp），进而得到82,829条单基因簇（unigenes）。单基因簇的长度范围为351 bp至28,928 bp，平均长度为1,155 bp。基于每个基因的每百万映射片段每千碱基转录本片段数（FPKM），采用DEGseq软件包，以Benjamini校正q值0.05作为筛选阈值，共鉴定出252个差异表达基因（differentially expressed genes, DEGs）。与未感染样本相比，ALV-J感染脾脏中共有90个基因呈上调表达，162个基因呈下调表达。
研究结论：本研究首次利用RNA测序技术在转录组水平解析了ALV-J感染鸡的脾脏组织。相较于未感染鸡，ALV-J感染脾脏中共发现252个差异表达基因，这些基因可作为后续探究ALV-J入侵机制的候选研究靶点。本研究通过Illumina Genome Analyzer IIx测序平台，完成了140日龄隐性白洛克鸡雌性个体的ALV-J感染组（WRR+）与未感染组（WRR-）的脾脏mRNA转录组测序。