Sample preparation – Different requirements for a wide ...€¦ · Sample preparation –...
20
Sample preparation – Different requirements for a wide range for genomic applications and platforms
Transcript of Sample preparation – Different requirements for a wide ...€¦ · Sample preparation –...
Sample preparation – Different requirements for a wide range for genomic applications and platforms
Where do you start?
• What genomic application?
• What technology?
• What platform?
• What sample type?
• How much sample is required?
• How to quantify for your sample type?
• What are the sample quality requirements?
• Do the samples need normalising/randomising?
• Are samples prepared with a control?
What genomic application?
CNV, InDels
Gene Expression
Methylation analysis SNP genotyping
miRNA expression
RN
AD
NA
What technology?
RT-PCR, qPCR
Microarrays
Sequencing
NGS, Sanger
Whole exome Genome
RNA-Seq
What platform?
What sample type?
Good Poor
Quantity ✓
Quality ✓
Blood
Good Poor
Quantity ✓
Quality ✓
Cells
Good Poor
Quantity ✓ ✓
Quality ✓
FFPE
Good Poor
Quantity ✓
Quality ✓
Plasma
DNA vs RNA
DNA RNA
• Stable• Robust• DNases less abundant in
standard lab environment than RNases
• Short term storage at -20°C• Ambient preparation
acceptable
• Inherently less stable than DNA
• Slightest exposure to RNase can impact RNA stability
• Short term storage at -80°C• On ice preparation required
Measuring quantity and quality
Absorbance
Bioanalyser
Fluorometric quantification
Limit of Detection
• NanoDrop: 2ng
• RiboGreen: 2ng
• Qubit: 250pg (20µL of sample required)
• Optical Density by SpectraMax: 5ng
• PicoGreen: 50pg
• RNaseP: 78pg
RNA integrityHigh quality intact RNA (9.7)
Heavily degraded RNA (2.5)Partially degraded RNA (7.7)
Normalisation, randomisation and controls
• Normalisation: Is normalisation necessary for your assay?Which dilution buffer do you use? DI water or TE buffer
• Randomisation: Eliminate bias by randomising your samples (i.e. batch, plate effects)
• Controls: Internal control, plate/batch control, processing control
Example case studies
Case study 1
• NSCLC patient samples
• 2,000 human FFPE samples
• RNA extracted (2/3 slides per sample)
• RNA concentration: Unknown
• RINs: Unknown
• Genome-wide expression analysis of protein encoding genes using Affymetrix Clariom S array
Case study 1What genomic application?
What technology?
What platform?
What sample type?
How much sample is required?
How to quantify for your sample type?
What are the sample quality requirements?
Do the samples need normalising/randomising?
Are samples prepared with a control?
Gene Expression
WT Microarray. Affymetrix Clariom S Array
Array plates on GeneTitan MC Instrustment
RNA from FFPE
WT Plus kit: 50-500ng RNAPico kit: 50ng-100pg RNA (non-FFPE), 500pg-50ng (FFPE) 3µL total Minimum: 167pg/µL
RNase P assay (optional: ND, RiboGreen, Qubit)
RIN using Bioanalyser. 18S qPCR
Yes. Randomise samples on 96 well plate
Internal controlProcessing plate control – leave 1 well empty per plate
Case study 2
• 24 human blood samples
• 4mLs of blood per sample
• Methylation analysis using Infinium Methylation EPIC arrays
Case study 2What genomic application?
What technology?
What platform?
What sample type?
How much sample is required?
How to quantify for your sample type?
What are the sample quality requirements?
Do the samples need normalising/randomising?
Are samples prepared with a control?
Methylation analysis
Infinium Methylation EPIC BeadChip array
Illumina iScan (16 samples per chip)
Bisulfite treated DNA
At least 650ng DNA in 5µL (130ng/µL)
Picogreen
260/280 Purity ratio between 1.8 - 2.0
Yes in TE buffer. Difficult as only 1.5 array chip
Internal control within kit reagents
Case study 3
• 96 mouse DNA samples, extracted from fresh tissue
• DNA concentration: All samples normalised to 40ng/µL 20µL total volume
• DNA purity: Purity ratios between 1.8 - 2.1
• Targeted sequencing AmpliSeq Comprehensive (>400 genes) Cancer Panel
Case study 3
What genomic application?
What technology?
What platform?
What sample type?
How much sample is required?
How to quantify for your sample type?
What are the sample quality requirements?
Do the samples need normalising/randomising?
Are samples prepared with a control?
Targeted DNA sequencing
Next generation sequencing using AmpliSeq Chemistry
Ion Torrent PGM/S3/S5
DNA
40ng in 5µL total volume (8ng/µL)
Nanodrop, Qubit or Picogreen
Intact DNA. 260/280 purity ratio between 1.8 - 2.2
Yes
Negative control
Summary
• Key considerations for sample requirements for genomic applications:
1. Sample type
2. Technology
3. Platform
• Appropriate quantity and quality is critical for sample preparation
• Additional considerations: normalisation, randomisation, controls
