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