The GSL offers long range genomic sequencing on the Chromium platform from 10X Genomics. Applications for this technology include generating phased genomic data, finding structural variants in human genome and exome samples, and assembly of genomes for novel organisms. Analysis of Chromium data is handled by several different software packages provided by 10X Genomics. First, a bclprocessor is used to generate specially formatted fastq files. Human samples may then be analyzed using Long Ranger to yield high-quality hapotype phasing and structural variant calls. Finally, results can be visualized using Loupe, a desktop genome browser. Supernova, an assembly tool for Chromium data, is available for de novo assemblies. If you are providing non-standard organism for Chromium library preparation, please include the estimated genome size in the Special Instructions. This information is required to titrate the input DNA. Software downloads are available on the 10X website.
Upon arrival to the GSL, all DNA samples are evaluated for concentration and integrity. DNA samples intended for the Chromium platform are evaluated with PicoGreen or Qubit and a 0.4% agarose gel with EtBr, or a pulsed field gel. A DNA sample intended for Chromium WGS or Exome protocol must be high molecular weight, which requires a specialized isolation procedure. If you are providing non-standard organism for Chromium library preparation, please include the estimated genome size in the Special Instructions. This information is required to titrate the input DNA.
Ideally, DNA should be above 50kb with greater than 80% of the DNA at 100kb or larger. After isolation, the DNA should be handled very gently (minimal pipetting, use of wide-bore tips, avoid freeze/thaw, etc). Ship HMW DNA on dry ice for stability, but once it arrives at the GSL it will be stored at 4C. Though the protocol uses 1ng of HMW DNA as input, a minimum of 500ng of DNA in 20ul of water or Tris should be submitted to allow for QC and visualization of the HMW DNA on a gel. The GSL can perform the HMW isolation if needed, and leftover DNA may be returned at the completion of the project.
Special note regarding isolation of HMW DNA from blood: a CPT blood collection tube with sodium citrate must be used for blood submissions (do not use the CPT tube containing heparin). After blood collection, follow the instructions provided with the tube for centrifugation to separate the blood into layers. Pull off the mononuclear cells and platelets and transfer to a new microfuge tube. The DNA from the cells can then isolated in your lab. If the GSL will perform the HMW isolation, spin the cells down into a pellet and remove as much buffer as possible. Freeze the pellet and ship it to us on dry ice.
The GSL currently offers:
Chromium libraries have a unique structure that require special sequencing considerations. Each DNA sample is indexed with four i7 barcodes sequenced as a standard i7 index read of 8bp in length. No i5 index read is sequenced for Chromium libraries. Instead, the unique barcode used to link reads is found at the beginning of read 1 and is 16bp in length. The GSL typically sequences Chromium libraries on the HiSeq X (150bp r1 / 8bp i7 / 150bp r2) or the HiSeq 2500 (125bp r1 / 8bp i7 / 125bp r2).
If Chromium libraries are returned to the customer for sequencing, the following information may be helpful for clustering. Chromium libraries are QC'd using qubit, bioanalysis, and kapa qPCR. For accurate nM determination by qPCR, the GSL adjusts the input fragment size and concentration (from flourmetric assays) by decreasing it 10-15%. In other words, reduce the fragment size by 10-15% (as determined by bioanalysis), reduce the concentration from Qubit by 10-15%, and use those values for the kapa qPCR calculation. This provides a more accurate nM concentration, prevents overclustering, and allows the samples to fall in the standard curve. This approach has been tested/validated within the GSL but your results and experiences may vary.