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Sample submission guide

To obtain the best sequencing data, the purity and size of the sequencing substrates are as important as the amount that is submitted.
We recommend users to QC their DNA samples prior to shipment and attaching the results (gel, trace analysis, concentration and ratios) to the sample submission form.

Please review our guidelines to make sure your sample is of sufficient quality to be sequenced.

DNA sample requirements
Essential requirements of your DNA sample:
- is double-stranded DNA, as single-stranded DNA is not compatible with the library preparation process.
- has not undergone multiple freeze-thaw cycles as they can lead to DNA damage.
- has not been exposed to high temperatures (e.g. > 37 °C for 1 hour), pH extremes (< 6 or > 9). Air drying of pellets is preferred to overheat drying. Avoid the over drying of genomic DNA. Do not heat when drying in a speed-vacuum.
- has an OD 260/280 nm ratio of 1.8 to 2.0 (measured at Nanodrop).
- does not contain insoluble material.
- does not contain RNA.
- has not been exposed to intercalating fluorescent dyes or ultraviolet radiation.
- does not contain chelating agents (e.g. EDTA), divalent metal cations (e.g. Mg2+), denaturants (e.g. guanidinium salts, phenol), or detergents ( e.g. SDS, Triton-X100).
- does not contain carryover contamination from the starting organism/tissue (heme, humic acid, polyphenols, polysaccharides, etc.).

Importantly, DNA should be shipped:
- in TE buffer (10 mM Tris-Cl, pH 8, 1 mM EDTA) for non-refrigerated international shipments
- in EB buffer (10 mM Tris, pH 8.5) for domestic (within the USA) and frozen (wet or dry ice) shipments.

Sample concentration should be 100 ng/μl or higher, the amount of the DNA should be at least 8 μg (for a single SMRT cell project).

 

DNA size profile and restriction enzyme accessibility:

DNA_QC_features_220630-A.png

A: Good DNA samples. Femto Pulse profiles of high quality, high molecular weight DNA, most of which are above 40 kb and very little at smaller sizes. Though with more DNA at 20-40 kb, the red sample DNA is still predominantly above 50 kb.

DNA_QC_features_220630-B.png

B: Acceptable DNA samples. Though the DNA molecules spread evenly between 10 and 165 kb, this DNA can still be used (with caution) in PacBio HiFi library prep.

DNA_QC_features_220630-C.png

C: Rejected DNA samples. The DNA of the blue and black samples is mostly below 10 kb – too small for a PacBio HiFi library. The red sample has a low amount and an irregular pattern indicative of issues with the sample or the DNA extraction.

DNA_QC_features_220630-D.png

D: RNase A and restriction enzyme digestion of HMW genomic DNA. The RNase A treatment removes transcripts, the DNA is accessible by endonucleases and is cut in fragments at a continuous size distribution.

 

RNA sample requirements
RNA samples should have a RNA integrity number (RIN) of 7 or more. Ideally, samples should have a RIN > 8.
Samples with RIN < 7 can still be processed, but the risk of significant underperformance or even failure is greater.
The ideal amount of total RNA is between 500 ng and 1 µg. The sample should be shipped in dry ice.

Sequencing of PCR amplicons
Products of PCR should be shipped upon in-house bead purification, in EB buffer and in a volume no larger than 47 µl.
The DNA amount varies depending on the amplicon size, and is reported in page 3 of this protocol.
Samples should be shipped in ice blocks or at a lower temperature.

 

 

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