Next-Gen Sequencing

 

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Transcriptome Sequencing
Introduction
A transcriptome is a collection of all the transcripts present in a given time point. The sequence of an RNA mirrors the sequence of the DNA from which it was transcribed. Consequently, by analyzing the entire collection of RNAs or transcriptomes, researchers can determine when and where each gene is turned on or off in the cells and tissues of an organism.
Whole Transcriptome Sequencing (WTS) or RNA sequencing can give researchers more visibility into changes that can occur in certain time points or states of diseases. Researchers can also see more details about how a transcriptome reacts to certain drugs, environmental situations or other conditions.
mRNA / mRNA + lncRNA Sequencing
This type of RNA sequencing is one of most popular methods for analyzing disease states and biological processes in the study of transcriptomes. This method is extremely in-depth, allowing researchers to find new genes that were previously imperceptible.
Library Preparation Methods
Enrichment
• Poly A enrichment (e.g. TruSeq Stranded mRNA)
• rRNA depletion (e.g. TruSeq Stranded Total RNA with Ribo-Zero)
cDNA Synthesis
• Random hexamer priming (e.g. TruSeq mRNA)
• Poly A priming (e.g. SMARTer Ultra Low Input RNA)
Strand Orientation
• Strand-specific approach provides gene expression information with relation to strand orientation.
Sequencing Platforms
• Illumina NovaSeq 6000 / HiSeq 4000 / HiSeq 2500
Data Analysis
Transcriptome de novo Sequencing
cDNA sequence reads are assembled into transcripts by a transcript assembly algorithm without the aid of a reference genome.
Standard Data Analysis
• Assembly
• Gene Expression Profile
Advanced Data Analysis
• BlastNT or BlastNR
• Differentially Expressed Genes (DEGs)
• Gene Annotations & Ontology Analysis
Transcriptome Resequencing
cDNA sequence reads are aligned to a reference genome. Availability of this analysis pipeline is dependent on the completeness of a reference.
Standard Data Analysis
• Alignment (Read mapping to current reference genome)
• Gene Expression Profile
• SNP calling (for Human, Mouse, Rat)
• Novel Transcripts
• Alternative Spliced Transcripts
• Fusion Gene (for Human, Mouse, Rat)
Advanced Data Analysis
• Differentially Expressed Genes (DEGs)
• Gene Ontology Analysis
• Customized Analysis
Small RNA Sequencing
This type of sequencing targets and isolates small RNAs (up to 200nt), including microRNA (21-23nt), by targeting the size range to discover how non-coding RNA affects different types of gene expressions. Small RNAs are known to be involved in regulation of gene expression in cellular process. Macrogen's NGS technology is able to sequence and quantify all the small RNA families in a sample and profile the miRNA, siRNA, piRNA and other non-coding RNAs.
Sequencing Platforms
• Illumina HiSeq 4000 / HiSeq 2500
Data Analysis
Standard Data Analysis
• Expression Profiles of small RNA (miRNA, tRNA, piRNA, snoRNA, snRNA etc.)
• Novel smRNA
• Categories of small RNA
Advanced data analysis
• Target Prediction of Known and Novel miRNA
• Gene Set Analysis
• Differentially expressed miRNA
Extracelluar RNA / Exosomal RNA Sequencing
Extracellular RNA or exosomal RNA, which exists outside cells in living fluids such as plasma or urine, can be used in various studies such as liquid biopsy through analysis of various transcription bodies (miRNA, piRNA, snRNA, snRNA, snRNA, and mRNA).
Sequencing Platforms
• Illumina HiSeq 4000 / HiSeq 2500
Data Analysis
Standard Data Analysis
• RNA composition analysis
• Expression profiles of smRNA and mRNA
• Novel miRNA prediction
Advanced data analysis
• Differentially expressed small RNAs
• Customized analysis
Iso-seq
By creating a full-length transcript, you can characterize the transcript isoform across the target gene or transcriptome. Specifically, it provides accurate information about the alternately spliced exon and transcription start site (TSS).
Library Preparation Kit
• PacBio Iso-Seq™ Express Template Preparation
Sequencing Platforms
• PacBio Sequel II / Sequel / RS II
Data Analysis
Standard Data Analysis
• Transcript isoform discovery and their validation
• Coding and annotation with SNPs
• Profiling and characterization junctions
• Quantitative determination and identification of regulatory transcripts
Advanced data analysis
• Differentially expressed small RNAs
• Customized analysis
*Data Analysis may vary depending on the availability of reference and the type of platform. For any inquiry of NGS service, please feel free to contact us.
E-mail : ngs@macrogen.eu