Library prep QC

Final QC

passed?

Sample A

Sample B

Water control A

Pool

Individual library QC passed?

Individual library QC passed?

Individual library QC passed?

YES

YES

YES

YES

QC 1

QC 2

Library prep QC

Concentration

Length

Remove adapter dimers

vs

vs

or

vs

Library prep QC - Concentration

Why is an accurate concentration important?

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• Individual samples: to pool equal amounts of different samples together
• Aim for an even amount of sequencing data per sample

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• Final library: sequencer requires loading with a very precise concentration
• If concentration is overestimated (actual conc. is lower than what you think)
• Underloading and underclustering of sequencing run
• Sequencing run will yield less sequencing data than usual

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• If concentration is underestimated (actual conc. is higher than what you think)
• Overloading and overclustering of sequencing run
• Sequencing run will suffer from higher error rates and reduced data output

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Library prep QC - Concentration

Spectrophotometry

(e.g. Nanodrop)

Fluorometry

(e.g. Qubit, qPCR)

• DNA absorbs light most strongly at 260 nm
• Level of absorbance ~ concentration of DNA

BUT other molecules (RNA, proteins) & contaminants also absorb light at 260 nm, causing up to 10-fold overestimation of the DNA concentration!

• Detects fluorescent dyes that only bind DNA
• Most reliable method for NGS

Electrophoresis

(e.g. Tapestation, Bioanalyzer)

• Compare intensity of DNA bands to standards
• Especially useful to determine concentration in certain fragment length range

DO NOT USE to quantify libraries

Library prep QC - Concentration

Qubit dsDNA High Sensitivity (HS) Assay kit

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1. Prepare Working Solution:
• Diluting HS reagent 1:200 in HS buffer

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• Dilute in Working Solution:
• Standards: 10 μl standard + 190 μl Working Solution
• Samples: 1 μl sample + 199 μl Working Solution

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• Vortex 2-3 seconds before running standards/samples

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• dsDNA HS Reagent (200x concentrate)
• dsDNA HS Buffer
• dsDNA HS Standard #1
• dsDNA HS Standard #2

Library prep QC - Concentration

Qubit dsDNA High Sensitivity (HS) Assay kit

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Select dsDNA HS workflow

Run the 2 standards

Enter sample volume added to tube

Run sample

Top value = original conc.

Bottom value = diluted conc.

Library prep QC - Concentration

• What if the concentration is too low?
• In future runs, consider adding more cycles to the indexing PCR step
• Amplify library using P5/P7 primers and qPCR-based library amplification kit
• e.g. Roche KK2702

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Standard 1

Standard 2

Standard 3

Standard 4

1. Set up qPCR reaction: library + mastermix + P5/P7 primers
2. Treat standards as separate samples (do not add mastermix)
3. Start run

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• Pause cycler during the 20s at 72°C step when a sample crossed the Standard 2 line, remove the sample and continue amplification for other samples
• After amplification, perform bead cleanup on amplified libraries and QC again

Library prep QC

Concentration

Length

Remove adapter dimers

vs

vs

or

vs

Library prep QC - Length

Why is it important to assess fragment length in a library?

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• Long fragments (>1.2 kb) do not cluster on Illumina flow cells and are therefore not sequenced

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• Short fragments are preferentially amplified (better clustering) and will thus be preferentially sequenced

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• Fragment length distribution impacts calculations to go from ng/μl (Qubit) to nM for loading concentrations

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Qubit

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Concentration in nM =

Capillary or gel electrophoresis

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Library prep QC - Length

Gel electrophoresis

Capillary electrophoresis

E.g. Tapestation, Bioanalyzer

1. PCR amplify an aliquot of the library using universal P5/P7 Illumina primers
2. Gel electrophoresis of ladder and samples

• Add buffer to aliquot of library
• Run machine

• Takes ~ 4 hrs

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• Tapestation ~ 10 minutes (depending on # samples)
• Bioanalyzer ~ 45 minutes
• No PCR bias
• Can calculate concentration in nM

Library prep QC - Length

Tapestation

• High Sensitivity D1000 or D5000 kits (1kb or 5kb detection limit)

Library prep QC

Concentration

Length

Remove adapter dimers

vs

vs

or

vs

Library prep QC - Clean up adapter dimers

• Adapter dimers are:

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• A byproduct of the adapter ligation step during library prep (TruSeq/NEBNext Ultra II DNA kits)

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• ± 140 bp long at the final QC step (after barcoding PCR)

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• Use capillary electrophoresis (preferred) or PCR + gel electrophoresis to detect adapter dimers

Adapter dimer

Adapter dimers

Library prep QC - Clean up adapter dimers

AMPure XP SPRI select beads (paramagnetic)

or homemade SPRI beads (>10x cheaper)

• The longer a fragment, the better it will bind to the beads
• The fewer beads you add, the more short fragments you’ll lose
• Perform size selection using these beads to remove adapter dimers

Volume beads

Volume reaction

SPRI ratio =

Example: for a 1.8x SPRI clean-up:

• Add 18 μl of SPRI beads to 10 μl of sample

Library prep QC - Clean up adapter dimers

AMPure XP SPRI select beads (paramagnetic)

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• Size selection using beads to remove adapter dimers

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• Rule of thumb: if you see a clear trace of adapter dimers, performs another round of cleanup

Library prep QC - Clean up adapter dimers

AMPure XP SPRI select beads (paramagnetic)

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​

​

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• Size selection using SPRI beads to remove adapter dimers

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• Rule of thumb: if you see a clear trace of adapter dimers, performs another round of cleanup

3 rounds

SPRI (0.85x)

After PCR amplification

After SPRI bead clean-up

ALL DIMERS SHOULD BE REMOVED!

Adapter dimers

Library prep QC

Final QC

passed?

Sample A

Sample B

Water control A

Pool

Individual library QC passed?

Individual library QC passed?

Individual library QC passed?

YES

YES

YES

How to fix samples with QC issues?

• Concentration too low -> amplify or restart
• Adapter dimers -> perform additional cleanups

NO

NO

YES

Fix it

Fix it