• Document: RNAseq Differential Gene Expression Analysis Report
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4553 Winters Chapel Rd Atlanta, GA 30360 (855) OTOGENE (686-4363) sales@otogenetics.com www.otogenetics.com RNAseq Differential Gene Expression Analysis Report Customer Name: Institute/Company: Project: NGS Data: IlluminaHiSeq2500 2x126bp PE Bioinformatics Service: Differential gene expression analysis Sample Species: Number of Samples: Date: Otogenetics Contact: Bioinformatics Phone: (855) OTOGENE (686-4363) Email: bioinfor@otogenetics.com Page 1 4553 Winters Chapel Rd Atlanta, GA 30360 (855) OTOGENE (686-4363) sales@otogenetics.com www.otogenetics.com 1. Description of Workflow Total RNA was submitted to Otogenetics Corporation (Atlanta, GA USA) for RNA-Seq assays. Briefly, the integrity and purity of total RNA were assessed using Agilent Bioanalyzer and OD260/280 using Nanodrop. 1-2 μg of cDNA was generated using Clontech Smart cDNA kit (Clontech Laboratories, Inc., Mountain View, CA USA, catalog# 634925) from 100ng of total RNA. cDNA was fragmented using Covaris (Covaris, Inc., Woburn, MA USA), profiled using Agilent Bioanalyzer, and subjected to Illumina library preparation usi ng NEBNext reagents (New England Biolabs, Ipswich, MA USA, catalog# E6040). 1.1 Illumina RNA-Seq sample preparation workflow Figure 1.1 RNA sample preparation. A. mRNAs are purified using Poly(A) selection from total RNA sample, and then fragmented. B. First strand of cDNA is synthesized using random priming, followed by the synthesis of the second strand of cDNA. C. The resulting double-strand cDNA from step B is end repaire d, phosphorylated and A-tailed. D. Adapter ligation and PCR amplification are p erformed, the library is ready for clustering and sequencing. Page 2 4553 Winters Chapel Rd Atlanta, GA 30360 (855) OTOGENE (686-4363) sales@otogenetics.com www.otogenetics.com 2. Raw Data Overview Table 2.1. Quality control. Data summary of generated reads . Ot## Ot## Ot## Ot## Ot## The quality, quantity and size distribution of the Illumina libraries were determined using an Agilent Bioanalyzer 2100. The libraries were then submitted for Illumina HiSeq2 500 sequencing according to the standard operation. Paired -end 90-100 nucleotide (nt) reads were generated and checked for data quality using FASTQC (Babraham Institute, Cambridge, UK). After achieving optimum QC results , samples were analyzed. Page 3 4553 Winters Chapel Rd Atlanta, GA 30360 (855) OTOGENE (686-4363) sales@otogenetics.com www.otogenetics.com 3. Bioinformatics analysis workflow To analyze the data we used the following pipeline. 3.1. Trimming sequence reads to remove adapters and low quality bases at ends 3.2. Fastqc quality control of Fastq file per sample 3.3. Mapping sequence reads to reference genome 3.4. Calculating reads count (FPKM) values of gene expression 3.5. Differential gene expression Analysis with unique read counts among samples Figure 3.1. RNA-seq workflow data analysis. Page 4 4553 Winters Chapel Rd Atlanta, GA 30360 (855) OTOGENE (686-4363) sales@otogenetics.com www.otogenetics.com 3.1. Trimming sequence reads to remove adapters and low quality bases at ends. Poor quality or technical sequences can affect the downstream analysis and data interpretation, which lead to inaccurate results. To assess quality of raw sequenced data, we used FastQC before and after trimming the adapters. Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality (error rate < 0.05) at the end. After

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