Linked Read Sequencing – 10X Genomics GemCode Technology

We are offering GemCode “linked read” library preps and sequencing. The sequencing libraries are generated with the GemCode instrument and reagents from 10X Genomics, followed by sequencing on our Illumina HiSeq sequencers.  When analyzing high molecular weight DNA samples the linked read sequencing data delineate linkage information over distances of up to 150 kb.  Just a few nanograms of sample arGemCodeBox390pxe required as input for the library preparation.

The applications currently supported by the 10X Genomics software are human haplotype phasing and structural variant detection (see this page for currently supported applications).  However, the technology is also valuable for de novo genome scaffolding, for structural variant detection in humans and other species, as well as for genotyping in repetitive regions of genomes (via an approach called “critical content rescue”). Please see these slides for details on GemCode technology and some of the possible applications.

The 10X Genomics technology generates individually barcoded sequencing libraries for hundreds of thousands of nanoliter volume oil droplets using up to 700,000 different barcodes. Thus it allows individual long DNA molecules to be analyzed by Illumina sequencing using the limiting dilution principle.  Please note that the individual DNA molecules are not meant to be fully assembled with this approach.  A single Hiseq lane generates sufficient information for phasing and structural variant analysis of a human sized genome.   Human phasing can be carried out using both whole genome shotgun sequencing as well as employing exome capture enrichment. 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.

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.  10X Genomics does suggest the Qiagen MagAttract HMW DNA Kit for DNA isolations (for plants other DNA extraction methods will be required).  Please see this suggested protocol.  The quality of the DNA samples should be verified by pulsed-field gel electrophoresis.  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).
The GemCode libraries do require paired-end 100 bp read sequencing – however, the first index read is unusually long (14 bases). Thus, these libraries will in most cases need to be sequenced in Rapid Mode on a HiSeq2500.

Before submitting samples for genome scaffolding: The DNA quality should best be verified by pulsed field electrophoresis. In the absence of PFGE equipment you could run the sample on a conventional agarose gel (0.7%) at low voltage together with a ladder containing a 20 kb band (e.g. GeneRuler 1 kb Plus)  and best also a lambda DNA sample (48.5 kb).  Please email us a gel image before shipping. Fluorometry (e.g. Qubit) should  be used for sample quantification and spectrophotometry should be used to assess the sample purity. Please see the sample requirements page.

Please inquire with us about the current sequencing recommendations and service recharge rates.