dna sequencing from single cell

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DNA sequencing from a single cell

Build a NGS library with comprehensive coverage and high sequence fidelity

https://www.qiagen.com/us/shop/sequencing/qiaseq-fx-single-cell-dna-library-kit/#orderinginformation

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Complete cell-to-library solution

PCR-free workflow

Primary sample isolation

Single cell isolation

NGS library constructionSample

• Single eukaryotic or bacterial cell• Picogram leves of purified DNA

• Whole genome NGS library◦ Illumina-compatible◦ Sequence variants◦ Structural variants◦ Aneuploidy◦ Bacterial genomes

InsightNGS run Data analysis Interpretation

QIAseq FX Single Cell DNA Library Kit

Single cell genomics by QIAGEN, 2016

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For single cell DNA sequencing

Ideally suited for• The analysis of inter-cellular genome heterogeneity• The analysis of aneuploidy & sub-chromosomal copy

number variations• Sequence variation analysis (SNV) in single cells• Whole genome sequencing from rare samples• Resequencing or de-novo sequencing of unculturable

microorganisms • New type of experiments, such as low-pass sequencing and

consensus-based variant calling

QIAseq FX Single Cell

DNA Library Kit


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Discover the QIAseq FX Single Cell DNA Library Kit

• Higher diversity libraries◦ PCR-free workflow, better library diversity by eliminating PCR duplicates

• Highest sequence fidelity◦ MDA-based amplification technology, proven for higher fidelity compared to PCR-based methods

• Complete genome coverage◦ Superior genome coverage and presentation of GC-rich regions

• Robust & streamlined workflow◦ Everything needed in one package, single use adaptors cut down on contamination possibilities◦ Under four hours workflow from single cell to library without any additional kits

• Enables Bio-Banking◦ Amplified DNA can be stored for follow-up experiments

MDA* instead of

PCR

Innovative QIAseq FX technology

Complete cell-to-library

solution

*MDA = multiple displacement amplification


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QIAseq FX Single Cell DNA Library Kit

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NGS library preparation for single cell DNA sequencing of eukaryotic or bacterial cells. Ideally suited for SNV or CNV analysis

• Robust and streamlined cell-to-library PCR-free workflow

• No PCR-duplicates - more diverse libraries

• Complete & uniform genome coverage, highest sequence fidelity and superior coverage of GC-rich regions

Superior genome coverage

Eliminate False Positives: PCR-free from 10ng cfDNABetter coverage of GC-rich regions Highest sequence fidelity


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QIAseq FX Single Cell DNA Library Kit: the contents

Both kits contain:• Cell lysis reagents

• Enzymes and buffers for whole genome amplification

• Enzymatic DNA fragmentation

• Single-step NGS library preparation

• Single-use, disposable Illumina Adapters in 96-well format

• Multiple reagent aliquots to reduce contamination risk and freeze-thaw cycles

What’s not included:• AMPure XP beads for library purification

• PCR reagents for library amplification are not needed as the entire workflow is PCR-free

• qPCR reagents for library quantification are recommended for accurate flow-cell loading

Cat No./ID: 180713QIAseq FX Single Cell DNA Library Kit (24)

Cat No./ID: 180715QIAseq FX Single Cell DNA Library Kit (96)


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QIAseq FX Single Cell DNA Library Kit: streamlined workflow

Cell lysis15 min

WGA2 h

FX library preparation

70 min

Purification20 min

ILLUMINA sequencing

3h 45 min h with ~40 min hands-on time

Cell Lysis• Start with single eukaryotic or

bacterial cells or small amounts (pg or ng) of intact gDNA

• Start with 4 µl cell material (1-100 cells) suspended in PBS (included)

• Prepare lysis buffer, mix with cells, incubate for 10 minutes at 65°C. Add 3 µl Stop Solution. If using purified DNA as input, incubate for 3 minutes at room temperature

• Hold at 4°C


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Cell lysis15 min

WGA2 h


70 min

Purification20 min

ILLUMINA sequencing


Whole Genome Amplification• Prepare WGA mastermix, mix with lysed cells, incubate for 2 h

◦ Amplified gDNA can be used directly or frozen until needed

◦ There will be an excess of amplified gDNA, which can be stored for later use or follow-up studies (e.g. confirming deletions detected with NGS via PCR or sanger sequencing)

◦ Library preparation accepts a wide range of inputs, so quantification of the amplified DNA is not needed


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Cell lysis15 min

WGA2 h


70 min

Purification20 min

ILLUMINA sequencing


NGS Library Preparation• Prepare FX mastermix, add to diluted WGA

product and incubate for ~15 min. Insert size can be set by user

• Hold at 4°C

• Add adapters from single-use adapter plate

• Prepare ligation mastermix, add to samples and incubate for 15 min to produce library


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Cell lysis15 min

WGA2 h


70 min

Purification20 min

ILLUMINA sequencing


Library Purification• Remove excess adapters with double-sided

Ampure XP cutoff

◦ No PCR amplification necessary; protocol generates sufficient library without enrichment

◦ Library quantification via qPCR (i.e. QIAseq Library Quant) is highly recommended to ensure accurate clustering on sequencer


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Discover complete genome coverage

Libraries were generated from single peripheral blood mononuclear cells (PBMCs) using the QIAseq FX Single Cell DNA Library Kit or kits from two other suppliers and sequenced at low depth using an Illumina MiSeq. Data were analyzed according to (1). Computed maximum achievable coverage by unlimited sequencing capacity are plotted.

Genome Coverage of various kits

(1) Zhang, C.-Z. et al. (2015) Calibrating genomic and allelic coverage bias in single cell sequencing, Nat. Comm. 6, 6822


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More uniform coverage, even in GC-rich regions

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Libraries were generated from either bulk gDNA using the QIAseq FX DNA Library Kit or from single peripheral blood mononuclear cells (PBMCs) using the QIAseq FX Single Cell DNA Library Kit or kits from two other suppliers.

Coverage versus GC content


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Highest sequence fidelity

Single cell libraries were prepared from isolated PBMCs using QIAseq FX Single Cell DNA Library Kit or kits from two other suppliers and sequenced with an Illumina MiSeq. Reads were mapped to the human genome (hg19) and sequence mismatches between NGS data and the reference were computed. All analyses were performed with CLC Genomic workbench 8.5.1. Data plotted are the mean proportion of sequence differences +/- standard deviation for 3 individual libraries prepared with each kit.

Error-rate of different single cell NGS methods


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Analyze copy-number variants and aneuploidy

Single cell libraries from PBMCs and Jurkat cell were prepared using QIAseq FX Single Cell DNA Library Kit and sequenced at a depth of 0.1x. Reads were mapped using BWA mem to human genome (GRCh38) and copy number variation of Jurkat vs PBMCs (control diploid cells) was assessed using the script published in (1). Plotted is the Log2 ratio (Jurkat/PBMC) of coverage using a window size of 500Kb for chromosome 2 from a cell with an approximately 25 Mbp deletion.

Detection of a 25 Mbp deletion in a single cell

(1) Chao Xie, Martti T Tammi, “CNV-seq, a new method to detect copy number variation using high-throughput sequencing”, BMC Bioinformatics, 2009,10:80, DOI: 10.1186/1471-2105-10-80


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High-yield WGA enables bio-Banking and confirmatory testing

Data from four individual PBMCs for each kit; with the kit from supplier R, no extra DNA was available for storage.

Yields from various kits


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Non-biased fragmentation for more uniform genome coverage

Single cell libraries prepared from PBMCs using QIAseq FX Single Cell DNA Library Kit or kits from two other suppliers were sequenced on an Illumina MiSeq. Plotted is the Nucleotide contribution of each Nucleotide (A, C, G, T) against the base position of the sequenced fragments.

Read start-bias of common single cell whole genome sequencing kits


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Robust FX accepts a wide range of inputs: save time on QC

For this experiment, we made libraries from different amounts of the same pool of amplified gDNA. Shown here are library yields after final purification. Libraries with a concentration of over 2 nM can be sequenced directly and don‘t need PCR amplification

Library yields from varying amounts of input WGA-DNA


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Highly Tunable Fragmentation: Determine Insert Size

We made libraries with varying amounts of amplified gDNA and ran a gradient of FX fragmentation times. The data show that the longer you incubate, the shorter the inserts get. This trend is relatively consistent regardless of the amount of input gDNA used (500-1500ng will be standard if the cells are in good condition and the instructions in the kit are followed, but quantification is not needed since the protocol is so robust)..

Mean insert size per incubation time


Download the DNA library prep product profile

https://www.qiagen.com/us/resources/resourcedetail?id=de78bde2-bdc5-46ae-892c-85d504b96c6f&lang=en

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Single cell genomics by QIAGEN

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For in-depth, molecular analysis of single cells

Single cell WGA or WTA

Single cell isolation

Single cell sequencing

• Easily access single cells

• Affordable

• Gentle on cells

QIAscoutQIAseq

FX Single Cell Library Kits

REPLI-g Single Cell Kits

Single cell miRNA analysis

• Create superior-quality NGS libraries directly from single cells

• Provides best-in-class amplification of genomes or transcriptomes from single cells

• Profile miRNA expression using qPCR

miScript Single Cell qPCR Kit


Visit the single cell resource center

https://www.qiagen.com/us/products/life-science-research/single-cell-resource/

dna sequencing from single cell

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