Next Generation Sequencing
Forms and Guidelines
Please read the sample submission guideline before submitting samples. It covers many aspects of sample preparation for NGS project and has become a useful toolkit for sample preparation and submission.
Samples are brought to the GSF (or are sent by an appropriate carrier for outside users) and are logged into Laboratory Information Management System (LIMS). We are currently using Wiki LIMS to keep track of sample receiving, QC, preparation and sequencing. Users are requested to submit the Sample Submission Form with the samples, with hard copy and electronic version when available. Samples then are quality control checked and the samples passing the QC will be moved forward to library production for sequencing purposes.
Sample submissions are initiated via entries in the LIMS database. After receiving samples, they are entered and recorded into LIMS. Every step involving the sample QC, library preparation, library quantification and sequencing is recorded and tracked in the LIMS. Users can also submit library to the GSF for sequencing. Users are requested to submit the Library Submission Form with the library, with hard copy and electronic version when available. Libraries then are quality control checked move forward to sequencing.
Please read through these important notes regarding to the sample preparation:
- Genomic DNA for DNA-Seq:
- Genomic DNA should be provided as high molecular weight DNA. Any sample exposed to phenol or other organic solvents should be run through a Qiagen cleanup column prior to submission to avoid contaminants that may inhibit the activities of enzymes used in the Illumina library preparation protocols.
- DNA samples should be treated with RNase.
- Preferred genomic DNA sample preparation methods include the Qiagen DNeasy kit and CTAB method.
- Please provide agarose gel picture of genomic DNA and readings from Nanodrop, Qubit or PicoGreen.
- Total RNA for RNA-Seq:
- For RNA-Seq sample, Trizol combined with Qiagen RNeasy mini kit should work well. Ambion’s mirVana miRNA isolation Kit, Qiagen’s miRNeasy kit, RNeasy kit, Tissue and lipid RNeasy kit, and microarray RNeasy kit are good options too. You will only need to stop at the total RNA isolation point when you use mirVana miRNA isolation Kit or Qiagen’s miRNeasy kit.
- Total RNA samples should be treated with DNase.
- Please provide Bioanalyzer trace file of total RNA and readings from Nanodrop, Qubit or RiboGreen.
- Total RNA for Small RNA-Seq
- We like to start with total RNA for small RNA–Seq application. We don’t recommend you enrich small RNA molecules from your total RNA extraction.
- For small RNA-Seq sample, Ambion’s mirVana miRNA isolation Kit and Qiagen’s miRNeasy kit is recommended to use for total RNA isolation. You will only need to stop at the total RNA isolation point when you use mirVana miRNA isolation Kit or Qiagen’s miRNeasy kit.
- Please provide bioanalyzer trace file of total RNA and readings from Nanodrop, Qubit or RiboGreen.
- ChIP DNA for ChIP-Seq:
- Users will need to think about the choice of antibodies, cell numbers and controls when they plan the experiment.
- Biological replicates are necessary, at least duplicate biological experiments should be done.
- Before ChIP, chromatin must be fragmented into manageable size. The optimal size range of chromatin for ChIP-Seq analysis should be between 150 and 300 bps. DNA fragments in this size range, which are equivalent to mono and dinucleosome chromatin fragments, provide high-resolution analysis of binding sites, and they work well for next-generation sequencing platforms.
- Please provide the agarose gel picture or Bioanalyzer trace file of ChIP-DNA and readings from Qubit or PicoGreen.
Following generation of the sequencing libraries, an aliquot will be run on an Agilent High Sensitivity DNA Bioanalzyer chip or Fragment Analyzer NGS kit to validate quality and size range of the library. The library will also be quantified by Qubit and qPCR to accurately measure the valid DNA template within the library. These steps enable us to load an appropriate quantity of the library on a flowcell to target optimal cluster passed filter rate.
Single Cell Analysis
Please note: 10X single cell service is by appointment only; we must have advance notice due to the nature of these preparations since we will need to have someone on standby to receive the cells. Please read 10x Genomics GSF Recommendations before you prepare/submit your samples to the GSF.
Please check these presentations if you are interested in this technology, sample preparation and data analysis.
When you consider preparing NGS library in your own lab and submitting library to GSF for sequencing with HiSeq 3000 system, please consider the following factors:
Library size restrictions
Libraries that are too long can result in polyclonal clusters that span more than 1 well, these will not pass filter. Smaller libraries will preferentially amplify with Illumina’s new kinetic exclusion amplification so tight library distributions ranging from 300-500 bp are recommended.
Very low tolerance for adapter dimers
Even as little as 1% adapter dimer can take up ~6% of sequencing reads, 10% contamination will take up 84% of reads. Illumina recommends you keep adapter contamination below 0.5% of your entire library.
Attn: Dawn Garcia
Greehey Children’s Cancer Research Institute, Room 2.120
UT Health San Antonio
8210 Floyd Curl Drive
San Antonio, Texas 78229