Thus nicks present in the DNA sample will significantly reduce the linked-read length. The first step of the actual 10X library prep is a denaturation of the sample. For DNA samples significantly longer than 50 kb these pre-treatments will likely not be necessary. For DNA samples at this size we might recommend two pre-treatments: DNA-Damage-Repair and BluePippin Sample Size Selection (the latter will remove fragments shorter than 40 kb). The DNA amount required for the actual library prep is minimal (1 ng), however more DNA is required for the QC of the sample (we suggest to submit at least 500 ng at a concentration of 20 ng/ul or higher as determined by Qubit).ġ0X Genomics recommends DNA sample should run as a band of 50 Kb length or higher on a pulsed-field gel. The quality of the DNA samples should be verified by pulsed-field gel electrophoresis. 10X Genomics does suggest the Qiagen MagAttract HMW DNA Kit for DNA isolations (for plants other DNA extraction methods will be required). The DNA quality will determine to a great extent the linkage information that can be gained from the 10X data – the longer the input DNA fragments the greater the length of the linkage information per droplet. The 10X Genome sequencing data contain conventional shotgun sequencing data, after trimming off the first 26 bases of the forward read. For human whole genome phasing and structural variant analysis GemCode sequencing data at 60x genome coverage or a combination of conventional Illumina data coverage at 30x genome coverage and GemCode at least 23x is recommended. Human phasing can be carried out using both whole genome shotgun sequencing as well as employing exome capture enrichment. A single HiSeq lane generates sufficient information for phasing and structural variant analysis of a human sized genome. Sub-genome sized parts of the DNA sample (about 1/6000th of a genome) are compartmentalized in nanoliter volume oil droplets together with beads carrying one of about two million different barcodes to initiate the library preparation with droplet specific barcodes. Please note that the individual DNA molecules are not meant to be fully assembled with this approach. DNA equivalent to about 150 copies of a genome does get distributed over about 1 million oil droplets that include beads. Thus, it allows individual long DNA molecules 10X Genomics Chromium Genome Linked-reads principle. The 10X Genomics technology generates individually barcoded sequencing libraries for hundreds of thousands of nanoliter volume oil droplets using up to 1.7 million different barcodes. Please see these slides for details on GemCode technology and some of the possible applications. Genotyping in repetitive regions of genomes is possible via an approach called “critical content rescue”. The applications currently supported by the 10X Genomics software are human haplotype phasing and structural variant detection (see this page for currently supported applications) as well as de novo genome assemblies (please see below). Just a few nanograms of sample ar e required as input for the library preparation. When analyzing high molecular weight DNA samples the linked read sequencing data delineate linkage information over distances of up to 150 kb. The sequencing libraries are generated with the Chromium controller instrument and reagents from 10X Genomics, followed by sequencing on our Illumina HiSeq sequencers. We are offering 10X Genomics Chromium Genome “linked read” library preps and sequencing. Please contact to reserve reagents for your project. As soon as these run out, we will discontinue the assay effective immediately. Update July 2020: We have ~16 remaining 10X Chromium Genome reactions left in our core. We are sad to see this very popular assay go. As of January 2020, 10X Genomics has announced that it is discontinuing production of the 10X Chromium Genome linked read reagents.
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