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

Sample to Insight

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2Complete cell-to-library solutionPCR-free workflow

Primary sample isolationSingle cell isolationNGS library construction

SampleSingle eukaryotic or bacterial cellPicogram leves of purified DNA

Whole genome NGS libraryIllumina-compatibleSequence variantsStructural variantsAneuploidyBacterial genomes

InsightNGS runData analysis

Interpretation

QIAseq FX Single Cell DNA Library KitSingle cell genomics by QIAGEN, 2016

Sample to Insight

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For single cell DNA sequencing3Ideally suited forThe analysis of inter-cellular genome heterogeneityThe analysis of aneuploidy & sub-chromosomal copy number variationsSequence variation analysis (SNV) in single cellsWhole genome sequencing from rare samplesResequencing or de-novo sequencing of unculturable microorganisms New type of experiments, such as low-pass sequencing and consensus-based variant callingQIAseq FX Single Cell DNA Library KitSingle cell genomics by QIAGEN, 2016

Sample to Insight

4Discover the QIAseq FX Single Cell DNA Library KitHigher diversity librariesPCR-free workflow, better library diversity by eliminating PCR duplicates

Highest sequence fidelityMDA-based amplification technology, proven for higher fidelity compared to PCR-based methods

Complete genome coverageSuperior genome coverage and presentation of GC-rich regions

Robust & streamlined workflowEverything needed in one package, single use adaptors cut down on contamination possibilitiesUnder four hours workflow from single cell to library without any additional kits

Enables Bio-BankingAmplified DNA can be stored for follow-up experiments

MDA* instead of PCRInnovative QIAseq FX technologyComplete cell-to-library solution*MDA = multiple displacement amplificationSingle cell genomics by QIAGEN, 2016

Sample to Insight

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QIAseq FX Single Cell DNA Library Kit5NGS library preparation for single cell DNA sequencing of eukaryotic or bacterial cells. Ideally suited for SNV or CNV analysisRobust and streamlined cell-to-library PCR-free workflowNo PCR-duplicates - more diverse librariesComplete & uniform genome coverage, highest sequence fidelity and superior coverage of GC-rich regions

Superior genome coverageEliminate False Positives: PCR-free from 10ng cfDNA

Better coverage of GC-rich regionsHighest sequence fidelitySingle cell genomics by QIAGEN, 2016

Sample to InsightMean coverage 0.1-0.2X 5

QIAseq FX Single Cell DNA Library Kit: the contents6

Both kits contain:Cell lysis reagentsEnzymes and buffers for whole genome amplificationEnzymatic DNA fragmentationSingle-step NGS library preparationSingle-use, disposable Illumina Adapters in 96-well format Multiple reagent aliquots to reduce contamination risk and freeze-thaw cycles

Whats not included:AMPure XP beads for library purificationPCR reagents for library amplification are not needed as the entire workflow is PCR-freeqPCR reagents for library quantification are recommended for accurate flow-cell loadingCat No./ID:180713QIAseq FX Single Cell DNA Library Kit (24)Cat No./ID: 180715QIAseq FX Single Cell DNA Library Kit (96)Single cell genomics by QIAGEN, 2016

Sample to InsightQIAseq FX Single Cell DNA Library Kit: streamlined workflow7Cell lysis15 minWGA2 hFX library preparation70 minPurification20 minILLUMINA sequencing

3h 45 min h with ~40 min hands-on timeCell LysisStart with single eukaryotic or bacterial cells or small amounts (pg or ng) of intact gDNAStart with 4 l cell material (1-100 cells) suspended in PBS (included)Prepare lysis buffer, mix with cells, incubate for 10 minutes at 65C. Add 3 l Stop Solution. If using purified DNA as input, incubate for 3 minutes at room temperatureHold at 4C

Single cell genomics by QIAGEN, 2016

Sample to InsightQIAseq FX Single Cell DNA Library Kit: streamlined workflow8Cell lysis15 minWGA2 hFX library preparation70 minPurification20 minILLUMINA sequencing

3h 45 min h with ~40 min hands-on timeWhole Genome AmplificationPrepare WGA mastermix, mix with lysed cells, incubate for 2 hAmplified gDNA can be used directly or frozen until neededThere 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 neededSingle cell genomics by QIAGEN, 2016

Sample to InsightQIAseq FX Single Cell DNA Library Kit: streamlined workflow9Cell lysis15 minWGA2 hFX library preparation70 minPurification20 minILLUMINA sequencing

3h 45 min h with ~40 min hands-on timeNGS Library PreparationPrepare FX mastermix, add to diluted WGA product and incubate for ~15 min. Insert size can be set by userHold at 4CAdd adapters from single-use adapter platePrepare ligation mastermix, add to samples and incubate for 15 min to produce librarySingle cell genomics by QIAGEN, 2016

Sample to InsightQIAseq FX Single Cell DNA Library Kit: streamlined workflow10Cell lysis15 minWGA2 hFX library preparation70 minPurification20 minILLUMINA sequencing

3h 45 min h with ~40 min hands-on timeLibrary PurificationRemove excess adapters with double-sided Ampure XP cutoffNo PCR amplification necessary; protocol generates sufficient library without enrichmentLibrary quantification via qPCR (i.e. QIAseq Library Quant) is highly recommended to ensure accurate clustering on sequencerSingle cell genomics by QIAGEN, 2016

Sample to InsightDiscover complete genome coverage11Libraries 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 Zhang, C.-Z. et al. (2015) Calibrating genomic and allelic coverage bias in single cell sequencing, Nat. Comm. 6, 6822Single cell genomics by QIAGEN, 2016

Sample to InsightComputed maximum genome coverage is plotted. This is the maximum achievable coverage of the genome, above which additional sequencing depth does not increase the proportion of the genome covered due to limited library complexity. The higher this number, the higher the proportion of the genome can be detected. This holds true regardless of sequencing depths, so regardless of the number of reads available, a library made with QIAseq FX SC DNA will cover more of the genome than a library prepared with competing methods.

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More uniform coverage, even in GC-rich regions12Libraries 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 contentSingle cell genomics by QIAGEN, 2016

Sample to InsightPCR based kits from supplier R and Y introduce GC bias and lead to underrepresentation of GC-rich regions. This can be extremely important since features of interest may be located in GC-rich stretches, or researchers may be working with bacterial samples with GC-rich genomes.

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Highest sequence fidelity13Single 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 methodsSingle cell genomics by QIAGEN, 2016

Sample to InsightHigher numbers mean a greater number of positions where the sequence in the dataset does not match the reference genome. Of course some of these sites will be normal polymorphisms or mutations, but many of these mismatches with either of the competing kits will represent false positives introduced during library preparation. These mismatches can increase background when calling variants, and can be identified as false positives in some cases.

Perfect for low-pass consensus variant callingLower background when analyzing sequence variants or mutations and small indelsFewer spurious sequence errors in your dataset, frewer false positives

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Analyze copy-number variants and aneuploidy14Single 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: 10.1186/1471-2105-10-80Single cell genomics by QIAGEN, 2016

Sample to InsightSingle cell libraries from PBMCs and Jurkat cells were prepared using the QIAseq FX Single Cell DNA Library Kit and were sequenced to 0.1x depth on a MiSeq.

Reads were mapped to human genome (GRCh38) and the copy number variation o