2) Next Generation Sequencing (NGS) - Sample Preparation

Summary

This video from abm provides a detailed overview of Next Generation Sequencing (NGS), focusing on sample preparation for various sequencing applications. It covers key techniques for preparing genomic DNA and RNA libraries, highlighting specific kits for Whole Genome Sequencing (WGS), Exome Sequencing (Exome-Seq), RNA Sequencing (RNA-Seq), and Methylation Sequencing (Methyl-Seq). The video explains the steps involved in library creation, validation, and sequencing, and discusses how to select the right kit based on DNA/RNA quantity and sequencing goals. For detailed protocols and tips, viewers are encouraged to explore abm's knowledge base.

Takeaways

• 😀 NGS sample preparation is a crucial first step before sequencing and involves creating a library from genomic DNA or RNA.
• 😀 The video covers various library preparation kits tailored to different sequencing applications, including WGS, Exome-Seq, RNA-Seq, and Methyl-Seq.
• 😀 Whole Genome Sequencing (WGS) requires 1-2 μg of DNA, with specific kits like TruSeq PCR-free, TruSeq Nano, and Nextera designed for different DNA quantities and genome complexities.
• 😀 The TruSeq PCR-free Library Prep Kit avoids PCR amplification errors, ensuring high accuracy for large genomes.
• 😀 The Nextera DNA Library Prep Kit speeds up sample preparation time for large genomes by using tagmentation, a process that both fragments and tags the DNA simultaneously.
• 😀 Exome Sequencing (Exome-Seq) is more affordable than WGS, as it focuses on sequencing only the coding regions (exons) of the genome, or optionally includes UTRs and miRNAs.
• 😀 The Nextera Rapid Capture Exome Kit is used for exome sequencing that targets only the exons, while the Expanded Exome Kit includes non-coding regions like UTRs and miRNAs.
• 😀 RNA-Seq focuses on sequencing RNA transcripts and can be done with specific kits like TruSeq Stranded Total RNA, TruSeq Stranded mRNA, or TruSeq Small RNA for different RNA types.
• 😀 Ribosomal RNA (rRNA) depletion is a key step in preparing total RNA for sequencing, as rRNA can dominate the RNA population, masking the signals from mRNA and other RNAs.
• 😀 The TruSeq Small RNA Kit is ideal for studying small RNAs, like miRNA, and involves unique procedures such as sequential adapter ligation and RT-PCR amplification.
• 😀 Methylation Sequencing (Methyl-Seq) is used to study DNA methylation, with a bisulfite treatment step that converts unmethylated cytosines to uracils while preserving methylated cytosines.

Q & A

What is Next Generation Sequencing (NGS)?

Next Generation Sequencing (NGS) refers to modern sequencing technologies that allow for high-throughput sequencing of DNA or RNA. NGS enables the sequencing of entire genomes, exomes, transcriptomes, and other biological sequences with high precision and speed.

What are the different types of NGS applications mentioned in the video?

The video mentions four main types of NGS applications: Whole Genome Sequencing (WGS), Exome Sequencing (Exome-Seq), RNA Sequencing (RNA-Seq), and Methylation Sequencing (Methyl-Seq). Each has specific uses for different types of biological analysis.

Why is PCR-free library preparation preferred for some WGS applications?

PCR-free library preparation is preferred for some Whole Genome Sequencing (WGS) applications because it avoids amplification errors that can occur during PCR. This ensures a more accurate representation of the genome, especially over long distances, as no amplification biases are introduced.

What is the main difference between TruSeq PCR-free Library Preparation Kit and TruSeq Nano DNA Library Prep Kit?

The main difference is that the TruSeq Nano DNA Library Prep Kit involves PCR amplification between adapter ligation and library validation, while the TruSeq PCR-free Library Preparation Kit does not use PCR. PCR amplification enriches the library in the Nano kit, which is helpful when only a small amount of DNA is available.

What is the purpose of the Nextera DNA Library Prep Kit in WGS applications?

The Nextera DNA Library Prep Kit is used in WGS applications to enable faster and more efficient library preparation. It uses a process called 'tagmentation,' where DNA fragmentation and adapter ligation happen simultaneously, reducing the time required for sample preparation.

What is Exome Sequencing and how does it differ from Whole Genome Sequencing?

Exome Sequencing (Exome-Seq) focuses on sequencing the coding regions (exons) of the genome, which make up about 2% of the whole genome. This is more affordable compared to Whole Genome Sequencing (WGS), which sequences the entire genome, including both coding and non-coding regions.

How does the Nextera Rapid Capture Exome Kit work for Exome Sequencing?

The Nextera Rapid Capture Exome Kit first tagmentates the DNA to add adapters, followed by PCR amplification. Afterward, exonic regions are captured using biotinylated probes, which specifically bind to exons. These regions are then enriched and purified for sequencing.

What is the difference between the TruSeq Stranded Total RNA Kit and the TruSeq Stranded mRNA Kit?

The TruSeq Stranded Total RNA Kit depletes ribosomal RNA (rRNA) and includes all RNA types in the analysis. In contrast, the TruSeq Stranded mRNA Kit focuses specifically on mRNA enrichment, allowing for a more targeted analysis of gene expression by removing rRNA.

What is the purpose of using the TruSeq Small RNA Kit for RNA sequencing?

The TruSeq Small RNA Kit is designed for the analysis of small RNAs, such as microRNAs (miRNA). The process involves adapter ligation to small RNAs, followed by RT-PCR amplification and gel isolation of the desired RNA products, ensuring a specific focus on small non-coding RNAs.

How does the TruSeq DNA Methylation Kit help analyze genome methylation?

The TruSeq DNA Methylation Kit analyzes genome methylation by treating fragmented DNA with bisulfite, which converts non-methylated cytosines into uracils while leaving methylated cytosines unchanged. This differential treatment allows researchers to identify and study DNA methylation patterns.