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).

A: Clamped Homogeneous Electric Field (CHEF) gel images showing genomic DNA of different size distributions (agarose gel, 1% TBE). Samples in lanes 1, 2, and 3 have little DNA below 30 kb, a uniform spread up to > 200 kb, and are considered good quality samples. Lane 4 has DNA that barely meets the minimum standard: a considerable amount of DNA below 30 kb, yet still a good amount of DNA at higher molecular weight. The sample in lane 5 has a DNA spread mostly between 15 to 60 kb and can still be used for sequencing. Samples in lanes 6 and 7 are too degraded – not good for sequencing. The DNA in lanes 8 and 9 does not move from the well, indicating that it may be complexed with other molecules (polysaccharides, proteins, etc.). Furthermore, the amount of contaminants prevents correct sizing and further processing. B: RNase A treatment and DNA restriction enzyme digestion test (agarose gel, 1% TAE). Samples 1 and 2 are of good quality: the RNase A treatment removes all the RNA, leaving a single band of unresolved high-molecular weight DNA. The digestion with restriction enzymes results in a continuous smear of small DNA fragments, proving that the sample is amenable to molecular reagents. Sample 3 has too little DNA to be quality checked and processed. Sample 4 has no RNA and is digested by the restriction enzymes - a good sample.

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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