MiSeq

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	==Next Generation Sequencing sample preparation==		==Next Generation Sequencing sample preparation==

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Revision as of 13:09, 15 January 2013

Contents 1 Next Generation Sequencing sample preparation 1.1 DNA sequencing 1.2 RNAseq 2 Useful links

Next Generation Sequencing sample preparation

DNA sequencing

Sample amounts

Genomic DNA >5ug (>200 ng/ul)

RNAseq

Sample RNA amounts

Total RNA >10ug (concentration: >200 ng/ul)

mRNA >1ug (concentration: >200 ng/ul)

Sample RNA quality

Intact rRNA should be observed in agarose gel electrophoresis of total RNA preparation

A260/280 ratio >1.8

A260/230 ratio >1.8

RNA purification

Commercial kits

Qiagen

Qiagen Total RNA extraction kits

Qiagen mRNA extraction kits - for eukaryotic mRNA only

Inivtrogen (Ambion)

RNA extraction

Useful links

• MiSeq Q&A
• NEBnext DNA library preparation kit
• UC Davis Genomic Center - Illumina Guide
• TCAG sequencing facility - following sample prep. guide...

The required amount of starting genetic material needed as recommended by the manufacturer will vary depending on the type of libraries and sequencing platforms. Below are some examples.

Genomic DNA for paired end sequencing: 500 ng to 5 ug.
Genomic DNA for mate-paired sequencing: 1 to 30 ug depending on insert size.
RNA-Seq or microRNA: 100 ng to 10 ug.
Exome sequencing: 6 ug.
ChIP-Seq: ≥ 20 ng.
Amplicon sequencing: ≥ 500 ng.
Library-ready aliquots: usually ¼ to half of the elution volume suggested in the manufacter's protocol.

Quantification of DNA or RNA by spectrophotometric methods may lead to overestimation of the concentration. 
If other methods such as Picogreen and qPCR are not available, we recommend sending twice the minimum amount required. 
Sample purity is important for a successful library preparation; please check that your sample has an OD260/280 between 1.8 and 2.0. 
For RNA samples, if possible check the integrity using an Agilent Bioanalyzer; the minimum RIN value acceptable for RNA library preparation is 8.
If a Bioanalyzer is not available, RNA integrity can be checked on a formaldehyde 1% agarose gel, or we can check on our Bioanalyzer on a fee-for-service basis.