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The Chromium™ System - Agilent...•Compound heterozygote is haplotype resolved for a 57kb...
Transcript of The Chromium™ System - Agilent...•Compound heterozygote is haplotype resolved for a 57kb...
The Chromium™ System
One System, One Workflow, Powerful New Sequencing Applications
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The Chromium™ System
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•Chromium™ Genome – The most comprehensive genome
–Long range genomes at population scale
–Call the full spectrum of variants and unlock previously inaccessible regions from a single library at equivalent coverage
•Chromium™ Exome – Reach beyond the exome
–Enable phasing of thousands of genes and detection of structural and copy number variation
–Optimized SureSelect baits to improve gene phasing by closing gaps, and recovering hard-to-map loci in the genome
•Chromium™ Single Cell 3’ – Cell-by-cell gene expression
–High-throughput single cell RNA sequencing
–Scalable transcriptional profiling of 1,000s to 10,000s of individual cells
Powerful new sequencing applications
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Complete solution from sample to answer with Chromium Exome and SureSelect baits
Sample input Target Enrichment Sequencers Analysis
Sample types• Cell lines• Demonstrated on
Whole blood and tissue
Sample prep methods• Qiagen MagAttract
HMW DNA kit
• Agilent All Exon V6 baits (available today)
• Agilent Optimized SureSelectBaits (2016H2)
• NextSeq• HiSeq 2000/2500• HiSeq 3000/4000
10x Software• Loupe 2.0• Long Ranger 2.0
Standard Outputs:• BAM• Fastq• VCF
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Partition Molecules and uniquely tag each short read to create Linked-Reads
Linked-Reads
• Input: Long DNA
• Output: Linked-Read data type enabling single molecule resolution
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GemCode™ Technology with millions of uniquely addressable partitions enables single molecule resolution and single cell sequencing
Conventional sequencing
100kb
Haplotype 1
Haplotype 2
Pos1 Pos2 Pos3
10x sequencing
Single Molecule ResolutionAverage of two molecules
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Mapping difficult in repetitive regions of genomes
Close Paralogs
Short Reads
Short Read Aligners Cannot Place Reads Correctly
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Long Range information Rescues Critical Content
1. Confident mapping provides anchors
2. Barcodes recruit short reads into paralogousregion
Close Paralogs
LariatTM Aligner Correctly Places Short Reads Even in Paralogous Loci
Linked-Reads
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Intelligent bait positioning improves phasing and mapping in difficult loci
Exome
Linked-Reads fromChromium Genome
Standard Exome Baits
Linked-Reads fromStandard Exome Baits
SureSelect OptimizedExome Baits
Linked-Reads fromOptimized Baits
Coming Q4 2016
Available Now
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10x Collaboration with Agilent Delivers High-Quality Constitutional Content
•Backbone: Agilent SureSelect Human All Exon V6
•Maximize ability to phase and call SV in all clinically relevant genes
•Optimize baits to improve mapping in clinically important genes –OPN1LW, OPN1MW, OPN1MW2
–SMN1, SMN2
–SRGAP2[ABCD]
Total design footprint: ~70 Mb
ClearSeq Inherited Disease2,874
TruSightOne4,811
MHC 26
ACMG56
257 10
2,194
Overlap of clinically relevant genes from various panels
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Similar coverage uniformity for 10x libraries vs standard libraries
• Chromium Exome (Agilent V6) library: 1.5 ng input DNA, 9 Gbsequencing
• Agilent SureSelect V6 library: 200 ng input DNA, 6 Gb sequencing
NA12878 Chromium Exome (9Gb)
vs. SureSelect (6Gb)
NIST Confident Regions
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Similar variant calling for 10x libraries vs standard libraries
• Chromium Exome (Agilent V6) library: 1.5 ng input DNA, 9 Gbsequencing
• Agilent SureSelect V6 library: 200 ng input DNA, 6 Gb sequencing
NA12878 Chromium Exome
(9Gb) vs. SureSelect (6Gb)
NIST Confident Regions
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• NA12878• RPS17: a ribosomal subunit gene in which mutations cause Diamond-Blackfan Anemia 4
Lariat Aligner: high quality mapping in the “dark”
BWA Aligner
Lariat Aligner
New TP variants
Same Data. Two Aligners. One Winner.
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Phasing of Compound Het in Titin with Chromium Exome
Confidential — Do not distribute
TTNCompound Het
189 kb (trans)
Work by: Daniel MacArthur
Mass General Hospital
“Seriously impressed with phased exome data from @10xgenomics -easily phases two causal TTN variants, 190 kb apart”
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• Autosomal recessive lysosomal storage disorder due to variants in CTNS
• Compound heterozygote is haplotype resolved for a 57kb deletion (exons 1-10 in CTNS) and a SNP
Compound Heterozygous Variant Resolution
chr17:3,563,221 (Gly308Arg)
CTNSSHPKTRPV1
57 kb deletion
G>A
Haplotype 1 Reads
Haplotype 2 Reads
Haplotype 1
Haplotype 2
NA17885 Chromium Exome
(SS v6) 9Gb
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•H2228: Non-small cell lung cancer
Identify Complex Structural Variants
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• VCaP: prostate cancer
Phasing Enables Detection of Single Exon Copy Number Changes
Copy Number Algorithms under active development
18• NA12214: Duplication of the PMP22 gene is associated with Charcot-Marie Tooth neuropathy type 1.
Larger CNVs are detected by read counts and classified/localized by Linked-Reads
CGH Confirmation using a 4x180k Agilent CGH array
CNV read count analysis of 10x libraries and SureSelect Exome plus phasing TE
The duplication is a tandem event, includes location and breakpoints
chr17:14,092,796
chr17:15,478,685
Copy 1 Copy 2
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Reaching Beyond Your Everyday Exome
Access genomic information from the Exome
Phase thousands of genes and assign cis or trans to heterozygous variants
Detect large SV and enable CNV detection
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10x Genomics products described are for Research Use Only. Not for use in diagnostic procedures.
Agilent products described are for Research Use Only. Not for use in diagnostic procedures.