Sample Input Guidelines
- Scott Patrick Hunicke-Smith
- Jessica Podnar
- Holly Stevenson
Sample Input Guidelines
NGS technologies and GSAF procedures can accept a wide range of sample amount and quality. By following these guidelines, you greatly reduce the risk of failure or poor results from sample-related issues, but successful sequencing results are routinely obtained for lower quantity or quality samples.
NOTE: The concentrations listed below should be determined by fluorometry. If you do not have access to a fluorometry-based assay (e.g. Picogreen/Qubit/etc.) and are relying on spectrophotometry, increase concentrations by at least ten-fold.
Please see notes below on guidelines for shipping tubes and plates
If you are submitting this... | Then submit in this... | For this platform... | GSAF requests this amount... | In this volume... |
|---|---|---|---|---|
| Finished NGS sequencing libraries | All Final libraries must be in 1.5 ml tubes | Illumina NovaSeq, NextSeq, MiSeq, or iSeq | >2nM | ≥20ul |
| DNA or RNA for QC | 1.5 ml tubes | Bioanalyzer-DNA | >50pg/ul | 3-5ul |
| Bioanalyzer-RNA Nano | 25-500ng/ul (Total RNA) 25-250ng/ul (mRNA) | |||
| Bioanalyzer-RNA Pico | .05-5ng/ul (Total RNA) .25-5ng/ul (mRNA) | |||
| Bioanalyzer-small RNA | 1-100ng/ul (Total RNA) 1-20ng/ul (enriched small RNA) | |||
| DNA - genomic DNA (NOTE: this assumes PURE POPULATIONS - i.e. 100% bacterial or fungal - you may need to assay samples with mixed populations prior to submission) | 96-well plates*, or 1.5mL tubes if you are submitting ≤12 samples | Metagenomics (bacterial or fungal) | 10ng/ul FOR ALL SAMPLES SUBMITTED (i.e. customer MUST normalize input samples) | ≥25ul |
| DNA - genomic DNA, high molecular weight, for library prep | 1.5 ml tubes, or 96-well plate for 12 or more samples* | DNA library prep, Covaris fragmentation | 1-80ng/ul | ≥25ul |
| DNA fragments (including IP-derived DNA) or DNA amplicons (PCR products), for library prep | 1.5 ml tubes, or 96-well plate for 12 or more samples* | DNA library prep | 1-80ng/ul | ≥25ul |
| RNA - total RNA, for library prep** | 1.5 ml tubes, or 96-well plate for 12 or more samples* | RNA-seq, PolyA purification | 10-80ng/ul | ≥25ul |
| RNA-seq, Ribosomal Depletion | 10-80ng/ul | ≥25ul | ||
| RNA-Seq, no removal or enrichment | 1-200ng/ul | ≥15ul | ||
| TagSeq | 25ng/ul (submitting RNA as low as 10ng/ul or as high as 100ng/ul is acceptable BUT ALL samples MUST be submitted at the same or very similar concentrations) Ideally you would submit at least 25ng/ul | ≥25ul | ||
| RNA-mRNA, for library prep | 1.5 ml tubes, or 96-well plate for 12 or more samples* | RNA-Seq | .2-20ng/ul | ≥15ul |
| RNA-miRNA or other RNA sub-fractions, for library prep | 1.5 ml tubes, or 96-well plate for 12 or more samples* | Small RNA library prep | 20-200ng/ul(Total RNA) 2-20ng/ul (purified small RNA) | ≥15ul |
* Samples should be plated starting at A1 then plate the samples vertically, A1 to H1, then A2 to H2 and so on.....Do not skip wells, you will be charged for wells that are skipped and we will not process samples that are plated A1 to A12. ** RNA must be free of DNA | ||||
Sample storage solutions
DNA or RNA can be provided in water, TE, low-TE, or other common storage solutions. If you are submitting your sample in something other than water or TE, please note this in either the Sample Description or Brief Job Description fields.
Marking your tubes and preparing to ship
Please write your sample names AND your job number ("JA20xxx") on your tubes before dropping off or shipping to the GSAF lab at MBB 3.210. You will not receive your job number until your electronic submission has been approved by the lab. For any jobs with DNA/RNA for library prep that will have 12 or less samples please submit in 1.5 mL Eppendorf lo-bind tubes, but any 1.5 mL tube is acceptable. Do not send 0.5 ml tubes. For DNA/RNA jobs that need library prep with greater than 12, the samples need to be submitted in a 96 well plate, when submitting your request please upload an excel file with a plate map into the supplemental data section.
If you are shipping your samples do not parafilm them if you have more than 8 tubes. If you have multiple samples we suggest shipping them in some type of box for storage, either plastic or cardboard. 50 ml conical tubes are also acceptable, please do not place your samples loosely among of box full of dry ice for shipment, they should always be in a secondary container.
Submitting Samples in 96 well plates (Required for large jobs)
Please be sure to write the job number on the side of the plate, if you are submitting multiple plates be sure to label them with a plate number, i.e. plate 1 JA20xxx. You will be required to submit a plate map that references wells to sample names, we ask that you include the actual well position in the sample name, i.e. A1_Test123, B1_Test567 ect...The plate map can be uploaded using the supplemental upload function when submitting the job.
Please use skirted or semi-skirted plates, and seal with a foil. Place a piece of cardboard or some protective barrier over the top of the foil for protection if shipping the samples. We would strongly recommend placing each plate inside its own box even if you are shipping multiple plates, be sure to thoroughly seal the plates!!
Samples should be plated starting at A1 then plate the samples vertically, A1 to H1, then A2 to H2 and so on.....Do not skip wells, you will be charged for wells that are skipped and we will not process samples that are plated A1 to A12.
The GSAF does not guarantee read counts but we will try our best to meet customer request when we prepare the libraries, however we will not make up any read counts for customer prepared libraries.
Always place tubes or plates in a secondary container, never ship samples loose in a box of dry ice!
Shipping
Shipping information is available here
Sample purity
We encourage you to evaluate purity (e.g. OD260/280 or OD230/260/280) as a measure of your nucleic acid extraction process. We do not provide guidelines for these measures because most of our NGS procedures begin with steps that are highly tolerant of contaminants, followed by purifications. If your samples fail our in-process QC while internal controls do not, we will contact you to troubleshoot further.
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