SAMPLE SUBMISSION GUIDELINES
Samples can be submitted in three different forms: raw material (tissue, cells, soil, etc.), purified nucleic acids (DNA or RNA), or prepared libraries¹. Please read carefully the quantity and quality requirements for each type of sample.
A failure to provide the required amounts and quality of sample material may render the performance of service(s) and/or order completion unattainable.
DNA/RNA Extraction Services
Details of Service:
1. All extracted DNA samples will be evaluated for quantity and quality with NanoDrop spectroscopy and/or Qubit fluorometry and/or agarose gel electrophoresis.
2. All extracted RNA samples will be evaluated for quantity and quality (RIN values) with NanoDrop spectroscopy and 2100 Bioanalzyer and RNA 6000 chip.
3. Any significant issues with sample quantity or quality shall be communicated to the individual requesting the service.
| DNA/RNA Extraction Services | Amount of Required Material* |
| DNA extraction -standard (blood, soft tissue, swabs, saliva, bacteria, yeasts) | 0.5 mL of blood; 20 mg of tissue, 2 swabs, 1 mL of saliva, 106 cells |
| DNA extraction -difficult (FFPE, bone, biofilm, soil, fungi spores, plants) | 5 slides, 100 mg of tissue, 50 mg of biofilm, 1 gm of soil, 100 mg of fungal material, 200 mg of plant tissue |
| RNA extraction – standard | 0.5 mL of blood; 20 mg of tissue, 2 swabs, 1 mL of saliva, 106 cells |
| RNA extraction – difficult | 100 mg of tissue; 50 mg of biofilm, 1 gm of soil, 100 mg of fungal material, 200 mg of plant tissue |
| RNA extraction FFPE | 6 slides or equivalent |
*The amounts identified above are the minimum requirements. We strongly recommend that your submission exceed these standards, if possible.
NGS Library construction from submitted nucleic acids (DNA or RNA)
Details of Service:
1. ACGT provides NGS library preparation services for Illumina® NGS platforms ONLY.
2. All DNA samples received for NGS library construction will be evaluated for quantity and quality with NanoDrop spectroscopy and/or Qubit fluorometry and/or agarose gel electrophoresis.
3. All RNA samples received for NGS library construction will be evaluated for quantity and quality (RIN values) with NanoDrop spectroscopy and 2100 Bioanalzyer with RNA 6000 chip.
4. Any significant issue(s) with sample quantity or quality will be communicated directly to the individual requesting the service.
5. At the request of the Sponsor, rRNA or globin RNA may be depleted from the samples.
6. DNA or RNA will be used to prepare single or dual-indexed libraries using the kits and/or methodologies appropriate for the specific service requested.
7. Appropriate quality control analysis will be performed at every step, and the libraries will be quantified via qPCR with the Kapa kit and/or Qubit fluorometer and/or the Agilent 2100 Bioanalyzer.
8. If multiplexed libraries are requested for a pooled HiSeq or NextSeq run, individual indexed libraries may be prepared as equimolar pools and loaded onto MiSeq™ flow cell to generate single-end 50 bp reads. The resulting information will be used to perform library evaluation and pooling adjustments. This step will be performed at the discretion of ACGT.
9. The requesting investigator will be provided with all results of library QC.
| NGS Library prep services | Amount of Material | Sample Quality Requirements | Fragment Size Range (bp) | Indexing |
| WGS (NexteraXT kit) | 100 ng | OD260/280 between 1.8 and 2.0;OD260/230 more than 1.6; concentration between 100 and 500 ng / µL | 200 – 600; 400 average | single or dual |
| WGS (Nextera) | 1,000 ng | 200 – 600; 400 average* | single or dual | |
| WGS (Nextera Mate Pair) | 5,000 ng | 500 – 5,000; 1,500 average | single or dual | |
| Exome (Agilent) | 6,000 ng | 50 – 300 | single or dual | |
| Exome (Illumina Rapid Capture) | 2,000 ng | 50 – 300 | single or dual | |
| Gene Panels (Illumina TruSeq / TruSight kits) | 2,000 ng | 50 – 300 | single or dual | |
| Amplicon (Illumina kits) | 100 ng / target | 50 – 300** | single or dual | |
| Amplicon (16S Metagenomics) | 200 ng | 500 | single or dual | |
| Amplicon custom-capture (HaloPlex, etc.) | 5,000 ng | various | single or dual | |
| Custom target libraries (Agilent SureSelect, etc.) | Variable (at least 6,000 ng) | 50-300; 150 average | single or dual | |
| ChIP-Seq (Illumina kits) | 200 ng | 30 – 100; 50 average | single | |
| RNA-seq (whole transcriptome) | 5,000 ng | OD260/280 more than 2.0;OD260/230 more than 1.8; concentration between 100 and 500 ng / µL; RIN>8.0 | 150 – 500; 300 average | single or dual |
| RNA-seq (small RNA) | 5,000 ng# | 25 – 75; 50 average | single |
*Average size of fragments prepared with Nextera kits can be increased upon request.
** Analysis of amplicons longer than 300 bp can be provided upon request.
# Submit 5 µg total RNA (or small RNA extracted from 5 µg total RNA) in 10 µl ultra pure water.
Sequencing of libraries using Illumina platforms
Details of Service:
1. ACGT provides NGS services using Illumina® platforms ONLY.
2. Please submit an aliquot of the stock library. We will evaluate and dilute it to an appropriate concentration.
3. All NGS libraries received for analysis on Illumina platforms will be quantified via qPCR with the Kapa kit and/or the Agilent 2100 Bioanalyzer and/or Qubit fluorometer.
4. Pooled multiplexed libraries will be loaded onto MiSeq™, NextSeq500™ or HiSeq4000™ flow cells to generate single or paired-end reads, and levels of coverage required for requested services.
Sample Submission requirements:
At least 10 µL per library is required, with a concentration between 5 and 20 ng/mL. Information regarding kits or methods used to prepare the libraries (including adapter and index sequences) must be provided.
Submission of incorrect information regarding the library adapter and index sequences material may render the performance of service(s) and/or order completion unattainable.