宏基因组学介绍--Illumina.pptxVIP

Metagenomics Sequencing:IntroductionFor Research Use Only. Not for use in diagnostic procedures.? 2020 Illumina, Inc. All rights reserved. Objectives● We will discuss the comprehensive workflow for ametagenomics sequencing project- What is metagenomics sequencing- Resources for getting started- Library preparation methods- Sequencing- Data analysis● This is an overview of many topics, so I will includehelpful resources along the way for more in depthinformation2 What ismetagenomicssequencing?3 Metagenomics sequencing● Metagenomics sequencing allows for the determination ofthe microbial diversity of a sample.● We may be looking at bacteria, fungi, archaea, viruses, orcombinations of these present in a sample.4 What starting material is used formetagenomics?● We start with isolated, purified DNA from our samplesource of interest.mixedsample- Sources include:- Soil- Skin swabs- Water- Plant surfaces- Etc.● Metagenomics samples may also be called mixedsamples, as they contain a mixture of different species.5 DNA input● It is very important to have highly pure DNA, free of anyinhibitors and contaminants.● When possible, use DNA purification kits developed forsample type.- Or a method described or validated for sample type● DNA/RNA isolation considerations for Illumina LibraryPreparation Kits- /bulletins/2016/05/dnarna-isolation-considerations-when-using-truseq-library-prep-kits.html6 DNA input● Input DNA must be accurately quantified, using afluorometric method for quantifying double strandedDNA, like Qubit or PicoGreen.● Control samples are optional, though can be helpful.- Commercial mock communities- Well-characterized samples7 What metagenomics is not● Another method commonly used with mixed samples ismetatranscriptomics, or microbial transcriptomics.● Metatranscriptomics is the sequencing of the RNApresent in a mixed sample.● More information on metatranscriptomics can be foundhere:- /areas-of-interest/microbiology/microbial-sequencing-methods/microbial-transcriptom