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High-throughput Sequencing Sample Submission Guidelines

DNA samples

DNA samples are recommended to be stored at -20℃. For short-term storage or transportation, DNA samples may be stored at 4℃ or transported using ice gel pack or ice packs. For long time transportation, dry ice and ice packs are recommended. DNA sample requirements are shown in Table 1.

Table 1. DNA sample requirements.

Sequencing type
Library type
Sample requirement
genomic sequencing Small fragment library ≥ 300ng, OD260/280: 1.8-2.2, concentration > 10ng/μl, volume > 10μl. The DNA bands of agarose gel electrophoresis are clear and no degradation, no RNA and protein contamination, and the samples are clear, colorless, sticky and soluble.
Pacbio Library (10/20K Library) ≥ 10μg, concentration > 100ng/μl, OD260/280 ratio: 1.8-2.0, OD260/230 ratio: 2.0-2.2. The ratio of Nanodrop concentration to Qubit concentration is ≤ 2, the sample is clear and colorless, and there is no insoluble matter; the agarose gel electrophoresis DNA band is clear and shows no DNA degradation, free of RNA and protein contamination.
Metagenome sequencing ≥ 300ng, OD260/280: 1.8-2.2, concentration > 2ng/μl, volume > 10μl. The agarose gel strips are clear and not significantly degraded.
16S/18S/ITS sequencing ≥ 15ul, the amplification result concentration ≥ 10nM and the genomic concentration ≥ 0.05ng/ul.
ChIP-Seq ≥ 30ng, concentration ≥1ng/μl, volume ≥10μl, peak shape is normally distributed between 100-500bp, with a main peak at 200-400bp. The sample is clear, colorless, non-viscous, soluble and free of protein contamination.
Whole genome methylation sequencing ≥ 1.5μg, the ratio of 260/280 is between 1.8-2.2, the concentration ≥ 20ng/μl, and the volume is greater than 10μl. The agarose gel electrophoresis band shows no significant degradation, and the sample is clear and colorless, non-viscous, and free of insoluble matter.
Exome sequencing Genomic sample, basically no degradation ≥ 300ng, 260/280 ratio between 1.8-2.2, concentration ≥ 2ng/μl, volume ≥ 10μl. The agarose gel electrophoresis band shows no significant degradation, and the sample is clear and colorless, non-viscous, and free of insoluble matter.
Special samples such as FFPE allow a slight degradation ≥ 400ng, the ratio of 260/280 is between 1.8 and 2.2, the concentration ≥ 4ng/μl, and the volume ≥10μl. The agarose gel electrophoresis band is above 500bp.
Amplicon sequencing Small fragment directly built library ≥ 150ng, 260/280 ratio: 1.8-2.2, concentration ≥ 2ng/μl, volume ≥ 10μl. The fragment size is between 100bp and 500bp, and the sample is clear and colorless, non-viscous, and free of insoluble matter.
Need to interrupt (segment size >1000bp) ≥ 500 ng, the ratio of 260/280 is between 1.8 and 2.2, the concentration is ≥ 20ng/μl, and the volume is ≥10μl. The agarose gel electrophoresis band is clear and not significantly diffused, and the sample is clear and colorless, non-viscous, and free of insoluble matter.
Library sample The concentration of the library ≥ 3nM (qPCR quantitative concentration), the concentration of the lane library ≥ 5nM (qPCR quantitative concentration), the volume ≥ 15μl. The sample contains no magnetic beads, and the library is compatible with the designated sequencing platform. Other notes:
1) The DNA library is dissolved in an appropriate buffer of Illumina Resuspension Buffer (RSB) or other buffer.
2) Clients should provide species information, fragment size,
database construction method, Agilent 2100 Bioanalyzer quality inspection chart and Qubit results.
3) HiSeq library size is between 300bp and 500bp, MiSeq library size is between 300bp and 600bp (no primer dimer and linker dimer).

RNA sample

RNA samples are recommended to be stored at -80°C. Dry ice or liquid nitrogen can be used for short-term storage or transportation. For long-term transport, it is recommended that the RNA be precipitated in 75% ethanol and transported in sufficient dry ice or blue ice (2 kg/day). RNA sample requirements are shown in Table 2.

Table 2. RNA sample requirements.

Sequencing type
Species type
Sample requirement
PolyA screening library Mammals ≥ 2μg, 260/280 ≥ 2.0, RIN > 7, 28S/18S ≥ 1.0, concentration > 50ng/μl, volume ≥ 10 μl, normal 5S peak. 2100 peak shape is normal, the sample is clear and colorless, no viscous, no insoluble matter.
Insects ≥ 2μg, 260/280 ≥ 2.0, concentration > 50ng/μl, volume ≥ 10μl, no rise (deletion) at baseline, normal 5S peak. 2100 peak shape is normal, the sample is clear and colorless, no viscous, no insoluble matter.
Plants, fungi, yeast, other animals ≥ 2μg, 260/280 ≥ 2.0, RIN > 6.5, 28S/18S ≥ 1.0, concentration > 50ng/μl, volume ≥ 10μl, 5S peak is normal. 2100 peak shape is normal, the sample is clear and colorless, no viscous, no insoluble matter.
rRNA removal library Eukaryotic organisms (lncRNA) ≥ 2μg, 260/280 ≥ 2.0, RIN > 7,23S/16S ≥ 1.0, concentration > 100 ng/μl, volume ≥ 10μl, 5S peak is normal.
Prokaryotes ≥ 2μg, 260/280 ≥ 2.0, RIN > 7, 23S/16S ≥ 1.0, concentration > 100 ng/μl, volume ≥ 10μl, 5S peak is normal. The sample is clear, colorless, non-viscous, and free of insoluble.
Small RNA sequencing Mammals ≥ 2μg, 260/280 ≥ 2.0, RIN > 8, 28S/18S ≥ 1.0, concentration > 170ng/μl, volume ≥ 10μl, normal 5S peak. The sample is clear, colorless, non-viscous, and free of insoluble.
Insects ≥ 2μg, 260/280 ≥ 2.0, concentration > 170 ng/μl, volume ≥ 10μl, no elevation at baseline, normal 5S peak. The sample is clear, colorless, non-viscous, and free of insoluble.
Plants, fungi, yeast, other animals ≥ 2μg, 260/280 ≥ 2.0, RIN > 7.5, 28S/18S ≥ 1.0, concentration > 170 ng/μl, volume ≥ 10μl, normal 5S peak. The sample is clear, colorless, non-viscous, and free of insoluble.
Macro transcriptome ≥ 3μg, concentration ≥ 100ng/μl, RIN ≥ 6.5, volume ≥ 10μl, the sample is clear and colorless, no viscous, no insoluble matter.
Full-length transcriptome sequencing 3-5μg or more, concentration ≥ 500ng/μl, 260/280 ≥ 2.0, RIN > 9, 23S/16S ≥ 1.0, the volume ≥ 10μl, and the 5S peak is normal.

Tissue sample

If DNA is to be isolated, tissue samples can be stored or transported for a short time, and stored or transported at 4 °C. For long-term transportation, dry ice and ice packs are recommended. If RNA is to be extracted, tissue samples should be transported using dry ice or liquid nitrogen. Please clearly mark the sample name, place the sample tube in a 50ml centrifuge tube or sample box, and then properly wrap it with foam or cotton to ensure that the sample is in good condition when it arrives at our laboratory.

Tissue sample requirements are shown in Table 3 and 4

  • DNA
    The DNA yields of different types of samples vary greatly. Please submit samples according to the actual conditions of the samples and the requirements of sequencing library construction. Tissue samples for DNA-related sequencing services should be shipped with ice packs.

Table 3. Tissue sample requirements for DNA sequencing.

Sample type
Sample requirement
Blood ≥ 2ml of fresh or cryopreserved anticoagulation (whole blood or white blood cells extracted from whole blood), EDTA anticoagulant is recommended, and heparin anticoagulation cannot be used to avoid affecting the experiment.
Isolated tissue ≥ 0.5 g of animal tissue, ≥ 2g of plant tissue.
Cell ≥ 5X106
Bacterial sample ≥ 1g of the bacterial body precipitate (about ≥ 10ml in the logarithmic growth phase of the bacterial liquid); Pacbio library: ≥ 15g of the bacterial body is required to precipitate (about ≥ 150ml in the logarithmic growth phase).
FFPE sample At least 10 slices without HE staining, the thickness of ≥ 5 microns, the area of the tissue on the slice should ≥ 25 square millimeters, of which the number of nucleated cells accounts for more than 80% of all cells, and the content of tumor cell tissue ≥ 70%.
16S/ITS/18S sequencing sample Precipitated quantity > 2g, volume > 10ml, which is specifically determined according to the abundance of the microorganism.
Metagenomic sample Environmental samples: 5-10g soil; 3-5g faeces; 5-10g sediment; 25ml solution.
Water: volume: 5-10ml, passed through a 0.22μm or 0.45μm pore size filter.
  • RNA
    The RNA yields of different types of samples vary greatly. Please submit samples according to the actual conditions of the samples, and the requirements of sequencing library construction. Tissue samples for RNA-related sequencing services are transported using dry ice or liquid nitrogen, unless otherwise specified.

Table 4. Tissue sample requirement for RNA sequencing.

Sample type
Sample requirement
Blood ≥5 ml of lymphocytes collected from whole blood. The amount of bird or fish samples can be reduced appropriately. Lymphocytes are recommended to be fully lysed with 20 volumes of TRIzol LS. Customers can also send whole blood samples frozen by liquid nitrogen. It is suggested to add 3 times volume of TRIzol LS lytic liquid to fresh blood and send it with dry ice. The customers should take the risk of sample extraction.
Fresh tissue ≥ 500mg. Or increase the sample quantity if sample containing muscle fiber or fat as it has a low RNA content.
≥ 1g of plant tissue. While for complex plant tissues containing polysaccharide polyphenols, please send large numbers of samples as possible. Fresh tissue is recommended to be wrapped in tin foil or packed in EP tubes after washing and frozen by liquid nitrogen within 3 min.
Cell ≥ 5X106. Please wash and precipitate the pellet, fully lysed with TRIzol and send it.
Bacterial sample ≥ 500mg of precipitation of fresh bacteria is quickly frozen by liquid nitrogen within 3min. Fungal samples are not recommended for preservation in TRIzol lysis.
High-throughput sequencing sample Customers collect blood PBMC cells; The total number of cells is between 1X106-5X106, full lysed with 1ml TRIzol; send it with dry ice.

Additional Readings

The Methods for DNA Extraction and Purification

Recovering Plasmid DNA from Bacterial Cultures without Using Kits

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