MiSeq

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Revision as of 05:57, 14 November 2012 (view source) Igchoi (Talk | contribs) (→Useful links) ← Older edit		Revision as of 07:01, 15 November 2012 (view source) Igchoi (Talk | contribs) (→RNAseq) Newer edit →
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-	:Intact rRNA should be observed in agarose gel electrophoresis total RNA preparation	+	:Intact rRNA should be observed in agarose gel electrophoresis of total RNA preparation
	:A260/280 ratio >1.8		:A260/280 ratio >1.8
	:A260/230 ratio >1.8		:A260/230 ratio >1.8

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Revision as of 07:01, 15 November 2012

Contents 1 Next generation sequencing 1.1 DNA sequencing 1.2 RNAseq 2 Useful links

Next generation sequencing

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

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.