QIAseq FastSelect – One-step, rapid removal of rRNA and/or ......Sample to Insight QIAseq®...

Sample to Insight

QIAseq® FastSelect – One-step, rapid removal of rRNA and/or globin mRNA

Jonathan Shaffer, Ph.D., [email protected] Director, Research & Development, NGS Assay Development

Sequence what matters

QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 1

Sample to Insight

Legal disclaimer

QIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease.

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.


Sample to Insight

Agenda

Background

What the product is and how it works

Performance and application data

Summary

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Sample to Insight

Agenda

Background



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Sample to Insight

Whole transcriptome stranded RNA NGS: What does it mean?

Gene A

Gene B

Transcript ATranscript B

Non-stranded RNAseq• Transcript orientation is not retained• You cannot say if it’s A or B

Stranded RNAseq• Transcript orientation is retained• You can say if it’s A, B or both!

Chromosome 1Plus strand

Chromosome 1Minus strand

Tip: Start with more than 100 ng of total RNA to reduce duplicates.


Sample to Insight

Library enrichment/depletion strategies: Whole transcriptome NGS

Enrichment or depletion is necessary to maximize reads from the RNAs of interest

• Typical RNA composition in a cell: >80% ribosomal RNA

• Highly abundant transcripts consume a lot of reads

• Enrichment or depletion is used to obtain more reads from the RNAs of interest, such as: ◦ mRNA ◦ lncRNA


rRNA

tRNA

miRNA

Small RNA

mRNA

lncRNA

Sample to Insight

Library enrichment/depletion strategies: Whole transcriptome NGS

Enrichment/depletion strategies

Strategy selection depends on the objective of your whole transcriptome sequencing.

mRNA

mRNAIncRNA

Poly(A) enrichment

rRNA depletion


rRNAtRNAmiRNASmall RNAmRNAlncRNA

Sample to Insight

rRNA depletion methods: Hybrid capture or RNase H

Pretreatment methodologies that take more than 2 hours and 29 steps

Pretreatment methodologies are time-consuming and arduous with a higher chance for sample loss.

Bind ssDNA probes to rRNA (6 steps)

Degrade rRNA using RNase H(6 steps)

Degrade DNA probes(6 steps)

RNA purification(11 steps)

Bead washing(12 steps)

Probe hybridization(5 steps)

Removal of rRNA(6 steps)

RNA purification(14 steps)

RNase H methodHybrid capture method


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QIAseq FastSelect: An unparalleled unwanted RNA removal solutionFastSelect: Only one step

Problem: rRNA is a significant NGS contaminant• 80–90% of RNA in samples

Existing depletions: Slow, tedious• Time required: 2+ h• Number of steps: Typically 29–37

QIAseq FastSelect: Revolutionary• Time required: 20 min• Number of steps: One• Robustness: >99% rRNA removal• Compatibility: Virtually any RNA library prep kit

FastSelect: 80% less time


Sample to Insight

Agenda

Background



Summary

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Sample to Insight

QIAseq FastSelect: Workflow and specifications

• What does it do? Removes cytoplasmic and mitochondrial rRNA and/or globin mRNA◦ Number of tubes: Two tubes (one rRNA and/or one globin mRNA)◦ Number of reactions: 24, 96 and 384

• Species: Human, mouse, rat and custom

• How does it work? Using a proprietary method, FastSelect is hybridized to the RNA sample and unwanted RNAs are eliminated from the library prep (no enrichment necessary)

• Sample compatibility: FFPE tissues, blood, biofluids, tissues (fresh/frozen), cell lines

• RNA input: 1 ng – 1 µg

• Tested kit compatibility: QIAseq Stranded Total RNA Lib Kit (QIAGEN Group), TruSeq® Stranded (Illumina, Inc), NEBNext® Ultra II Directional (New England Biolabs, Inc), KAPA® RNA HyperPrep (Roche Group)◦ FastSelect is compatible with most RNA library prep kits

• Custom: Expandable to any species and any RNA

Stepwisecool from

75°C to 25°C (20 min)

Complete remaining

library preparation

steps

Perform fragmentation

(optional)

Combine QIAseq

FastSelect with RNA sample


Sample to Insight

Agenda

Background



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QIAseq FastSelect vs. no treatment, Poly-A and Ribo-Zero Gold

Experimental overview• Sample: 100 ng Universal Human Reference RNA (UHRR)• Enrichment: Poly-A (+mRNA), Ribo-Zero (-rRNA), FastSelect (-rRNA)• Library prep: QIAseq Stranded Total RNA Lib Kit• Sequencing: NextSeq® 550 (2 x 75 bp)• Mapping: CLC Biomedical Workbench

FastSelect efficiently removes rRNA to levels observed with Poly-A enrichment without disturbing protein coding genes.

Percentage of rRNA reads


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QIAseq FastSelect vs. No treatment, Poly-A and Ribo-Zero Gold

FastSelect vs. Ribo-Zero GoldGene expression

FastSelect vs. no treatmentGene expression

Poly-A vs. no treatmentGene expression

FastSelect does not perturb the natural expression profile of samples.

Results• FastSelect vs. no treatment: Highly similar results (a low number of expressed genes was

observed with “no treatment”, because of the high rRNA percentage, highlighting the need for rRNA depletion)

• FastSelect vs. Ribo-Zero Gold: Highly similar results, suggesting FastSelect does not have any spurious off-targets

• Poly-A vs. no treatment: Highly dissimilar results suggesting that poly-A enrichment may disturb the full expression profile of samples


Sample to Insight

QIAseq FastSelect: Robust performance with FFPE samples

Experimental overview

FastSelect robustly removes rRNA and enables high reproducibility of experiments. Ribo-Zero was not as effective with FFPE samples and required more amplification, suggesting loss of sample material.

FastSelect Ribo-Zero Gold

Replicate samples: Gene expression

Percentage of rRNA Reads

• Sample: 100 ng normal and cancer lung FFPE• Enrichment: FastSelect, Ribo-Zero Gold• Library prep: QIAseq Stranded Total RNA Lib Kit• Sequencing: NextSeq 550 (2 x 75 bp)• Mapping: CLC Biomedical Workbench


Sample to Insight

QIAseq FastSelect: Consistent and reproducible performance (SEQC)


• Sample: 100 ng SEQC samples• Enrichment: FastSelect• Library prep: QIAseq Stranded Total RNA Lib Kit• Sequencing: NextSeq 550 (2 x 75 bp)• Mapping: CLC Biomedical Workbench


PCA plot of SEQC samples

FastSelect results in highly efficient removal of rRNA from the SEQC samples. PCA of expression profiles demonstrates that replicate samples and the four reference samples cluster as expected.


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QIAseq FastSelect: Consistent performance, regardless of tissue


FastSelect efficiently removes rRNA from vastly different tissue types.

• Sample: 100 ng samples of adipose, adrenal gland, kidney, liver, lung, skeletal muscle and thyroid tissue• Enrichment: FastSelect• Library prep: QIAseq Stranded Total RNA Lib Kit• Sequencing: NextSeq 550 (2 x 75 bp)• Mapping: CLC Biomedical Workbench



Sample to Insight

Agenda

Background



Summary

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Sample to Insight

FastSelect is compatible with the QIAseq Stranded Total RNA Lib Kit

Other compatible kits:• TruSeq Stranded (Illumina)• NEBNext Ultra II Directional (NEB)• KAPA RNA HyperPrep (Roche)

FastSelect is compatible with most RNA library prep kits.


Sample to Insight

QIAseq FastSelect: An unparalleled unwanted RNA removal solution

20

FastSelect

Hottest (one-step method)

Easiest (handling)

Strongest (rRNA

removal)

Biggest (any

species and target)

Most flexible (sample type and amount)

Fastest (speed)

QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA

Sample to Insight

Thank you for attending!

Questions?

Jonathan Shaffer, Ph.D., MBA

[email protected]


Date post:	15-Oct-2020
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QIAseq FastSelect – One-step, rapid removal of rRNA and/or ......Sample to Insight QIAseq®...

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