Page 1
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
Page 2
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.
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 2
Page 3
Sample to Insight
Agenda
Background
What the product is and how it works
Performance and application data
Summary
1
2
3
4
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 3
Page 4
Sample to Insight
Agenda
Background
What the product is and how it works
Performance and application data
Summary
1
2
3
4
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 4
Page 5
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.
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 5
Page 6
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
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 6
rRNA
tRNA
miRNA
Small RNA
mRNA
lncRNA
Page 7
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
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 7
rRNAtRNAmiRNASmall RNAmRNAlncRNA
Page 8
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
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 8
Page 9
Sample to Insight
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
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 9
Page 10
Sample to Insight
Agenda
Background
What the product is and how it works
Performance and application data
Summary
1
2
3
4
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 10
Page 11
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
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 11
Page 12
Sample to Insight
Agenda
Background
What the product is and how it works
Performance and application data
Summary
1
2
3
4
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 12
Page 13
Sample to Insight
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
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 13
Page 14
Sample to Insight
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
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 14
Page 15
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
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 15
Page 16
Sample to Insight
QIAseq FastSelect: Consistent and reproducible performance (SEQC)
Experimental overview
• 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
Percentage of rRNA reads
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.
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 16
Page 17
Sample to Insight
QIAseq FastSelect: Consistent performance, regardless of tissue
Experimental overview
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
Percentage of rRNA reads
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 17
Page 18
Sample to Insight
Agenda
Background
What the product is and how it works
Performance and application data
Summary
1
2
3
4
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 18
Page 19
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.
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 19
Page 20
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
Page 21
Sample to Insight
Thank you for attending!
Questions?
Jonathan Shaffer, Ph.D., MBA
[email protected]
QIAseq FastSelect – One-step, rapid removal of rRNA and/or globin mRNA 21