Next Generation Sequencing
Forms and Guidelines
Please read the sample submission guidelines (update 8/5/25) before submitting samples. It covers many aspects of sample preparation for the NGS project and has become a valuable toolkit for preparation and submission.
Samples are brought to the GSF (or are sent by an appropriate carrier for outside users) and are logged into the Laboratory Information Management System (LIMS). We currently use Wiki LIMS to track sample receiving, QC, preparation, and sequencing. Users are requested to submit the SAMPLE Submission Form along with the samples, in both hard copy and electronic versions when available. Samples are then quality control-checked, and those that pass 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, from sample QC to library preparation, library quantification, and sequencing, is recorded and tracked in the LIMS.
Users can also submit the library to the GSF for sequencing. Users are requested to submit the LIBRARY Submission Form to the library, with a hard copy and an electronic version when available. Libraries are then quality-controlled and moved forward to sequencing.
Quick Links
Please read through these critical notes regarding the sample preparation:
In order for us to provide you with high-quality data, please review the following information for details on how to submit samples to the GCCRI Genome Sequencing Facility.
The sample submission form must accompany all submitted samples (Sample Submission Form). You must fill out all the information. Please note: The Sample_Name field entry in the sample submission form cannot contain illegal characters not allowed by file systems. Sample name must be unique, must not be longer than 20 characters. Permissible characters are only letters, numbers and underscores. Examples of common characters not allowed are the space character and the following:? ( ) [ ] / \ = + < > : ; ”
Please label each sample individually and carefully to make sure we can recognize all your samples. Please include a sample submission form in your sample package. DNA form samples should be on ice or dry ice, and RNA form samples should be in a dry ice package. For any project with DNA/RNA for library prep that has 12 or fewer samples, please submit in a 1.5 mL Eppendorf tube. Do not send 0.5 ml tubes. For any project involving DNA/RNA that requires library preparation with more than 12 samples, the samples must be submitted in 8 or 12 PCR strip tubes or a 96-well plate, labeled & appropriately sealed. Please do not place your samples loosely among a box full of ice or a dry ice package; they should always be in a secondary container such as a box, bag, or tube.
- 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 before 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 methods for genomic DNA sample preparation include the Qiagen DNeasy kit and the CTAB method.
- Please provide an agarose gel picture of genomic DNA and readings from Nanodrop, Qubit, or PicoGreen.
- Total RNA for RNA-Seq:
- For the RNA-Seq sample, a combination of Trizol and the 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 the mirVana miRNA isolation Kit or Qiagen’s miRNeasy kit.
- Total RNA samples should be treated with DNase.
- Please provide a 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 applications. We don’t recommend you enrich small RNA molecules from your total RNA extraction.
- For small RNA-Seq samples, Ambion’s mirVana miRNA isolation Kit and Qiagen’s miRNeasy kit are recommended for total RNA isolation. You will only need to stop at the total RNA isolation point when you use the mirVana miRNA isolation Kit or Qiagen’s miRNeasy kit.
- Please provide a Bioanalyzer trace file of total RNA and readings from a Nanodrop, Qubit, or RiboGreen instrument.
- ChIP DNA for ChIP-Seq:
- Users will need to consider the choice of antibodies, cell numbers, and controls when planning the experiment.
- Biological replicates are necessary; at least, duplicate biological experiments should be done.
- Before ChIP, chromatin must be fragmented into a manageable size. The optimal size range for chromatin in ChIP-Seq analysis is between 150 and 300 base pairs (bp). DNA fragments in this size range, equivalent to mono- and dinucleosome chromatin fragments, provide high-resolution analysis of binding sites and are well-suited for next-generation sequencing platforms.
Please provide the agarose gel picture or Bioanalyzer trace file of the ChIP-DNA, along with the corresponding readings from Qubit or PicoGreen.
Single Cell Analysis
Please note: 10X single-cell service is by appointment only. We require advance notice due to the nature of these preparations, as we will need to have someone on standby to receive the cells. Please read the 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.
Users on campus can drop off the samples (DNA form samples should be on ice or dry ice, and RNA form samples should be on dry ice). GCCRI building address:
Genome Sequencing Facility
Room 2.120
Greehey Children’s Cancer Research Institute
8403 Floyd Curl Drive, San Antonio, Texas 78229
Attn: Korri Weldon
Phone 210 562-9125
Outside users will need to ship their samples with enough dry ice via FedEx overnight shipping. Here are the shipping instructions:
Please include a sample submission form in your shipping package. Samples shipped to the GSF should be sent in 1.5 mL tubes, PCR strip tubes, or 96-well plates and labeled & sealed well. Please do not place your samples loosely among a box full of dry ice for shipment; they should always be in a secondary container such as a box, bag, or tube. If shipping plates, each plate should be carefully packed. We also recommend that all shipments be made on dry ice and that samples M-W be shipped overnight. Please see our Guidelines for Submitting Samples for sample input information.
The GSF shipping address is:
Attn: Korri Weldon
Greehey Children’s Cancer Research Institute Room 2.120
UT Health San Antonio
8403 Floyd Curl Drive
MC7784
San Antonio, Texas 78229
Phone 210 562-9125
DOWNLOAD: Single-Cell Gene Expression User Sample Table (XLS)