10X Chromium Genome
The 10X Chromium Genome Solution allows long-range sequence information to be generated from a short-read Illumina sequencer. The Chromium Genome Solution employs the power of Linked-Reads to resolve geneic phasing, reveal structural variation and detect variants in complex regions of the genome. With Linked-Read data, investigators can call and phase major classes of structural variants (SVs) like deletions, inversions and translocations. Phasing of single nucleotide variants (SNVs), indels and SVs can be performed accross >10 Mb haplotype blocks.
Research areas supported by the 10X Genome Solution
• Cancer Research: Determine the molecular basis of cancer with better understanding of tumor heterogeneity by discovering more variants in a single assay.
• Genetic Health: Improve the understanding of disease inheritance by unlocking the full spectrum of variants in a single workflow.
• Population Genetics: Attain the most comprehensive view of variants and access more disease-associated regions.
• Exome Interrogation: Enrich Linked-Read Genome libraries with targeted baits to obtain a complete exome assay.
Powered by 10X GemCode Technology, the Chromium Genome Solution partitions and moleculary barcodes gDNA on the 10X Chromium Controller to produce sequence ready, Illumina compatible libraries with >1,000,000 unique barcodes. The Chromium Software Suite contains the Long Ranger and Loupe Genome Browser software packages which provide turn-key analysis pipelines and visulaization tools for Linked-Read genome data.
Linked-Read Genome Libraries prepared using the 10X Chromium Controller are Illumina compatable and will be sequenced using a 150 paired end (PE) sequencing strategy, targeting 425 M PE reads per library on the NovaSeq 6000. A 100 PE sequencing strategy is used to target 9 Gb of sequence coverage for exome enriched libraries using the Agilent SureSelect Human All Exon v6 exome capture.
The success of a 10X Genome Solution project is determined by the purity and integrity of the high molecular weight (HMW) genomic DNA (gDNA) provided to the Genomics Core. All HMW gDNA submitted for 10X Genome sequencing library preparation or exome enrichment will be assayed on the Agilent TapeStation using the Genomic DNA Sceen Tape assay to assess sample integrity (DNA Integrity Number - DIN) and verify the HMW gDNA is ≥ to 50Kb in size. The optimal size is ≥ 65Kb which can maximize haplotype phase block length and increase the ability to call SVs. The gDNA must meet purity standards with a 260/280 ratio between 1.8 - 2.0 and 260/230 ratio at ~2.0. The Genomics Core - Microarray Facility offers a HMW gDNA isolation service using the QIagen MagAttract HMW DNA kit which produces ≥60kb gDNA isolates from cells and tissue.
Best Practices for Handling HMW gDNA
• Provide ~250 - 500 ng of HMW gDNA @ 50 - 100ng/ul.
• Isolate gDNA from fresh cells or tissue, when possible, using a kit or method capable of obtaining >50Kb HMW gDNA.
• Use nuclease-free water or elution buffer (EB buffer) to resuspend gDNA. Avoid EDTA containing eluants. Use nuclease-free consumables.
• Never vortex tubes containing HMW gDNA. Use 50ºC block or water bath and gentle finger mixing to fully dissolve the gDNA if needed.
• Use wide-bore 200ul pipet tips when manipulating isolated HMW gDNA.
• Extracted HMW gDNA samples (>10ng/ul) can be stored at 4ºC for up to 2 weeks or at -20ºC for up to 6 months.
• Prepare multiple small aliquots for -20ºC storage to avoid repeated freeze thaw cycles of HMW gDNA.