Sample requirements

General guidelines for all applications:

  • Important! If the sample requirements are not followed (with too low concentration and/or volume), the consequence could be less complex libraries and negatively affected data quality.
  • Provide the recommended amount if possible, which allows for two library preparations in case the first prep fails for any reason.
  • If providing a lower amount than the recommended, always make sure to provide more material than the sample requirement to ensure enough material for QC and prep.
  • If a submitted sample fulfills the requirements but analysis fails, we will do a second attempt to analyze the sample (granted that there is enough material). If the second analysis also fails, we will invoice the samples. This is not applicable to the microbial application MWXNXxR002, see section "Whole genome sequencing of microbial DNA" below.
  • If providing lower amount than the sample requirement, please contact Clinical Genomics prior to sample submission.

Table of contents:


Panel- and Whole Exome sequencing

Amount:

Important! If the sample requirements are not followed (with too low concentration and/or volume), the consequence could be less complex libraries and negatively affected data quality.

DNA concentration needs to be measured with fluorometric measurements e.g. Qubit (Invitrogen), not with absorbance measurements.

Submit 20 - 130 ul. However, ensure that the total amount of DNA is sufficient (see below).

Note! If possible, provide the recommended amount which allows for two library preparations in case the first prep fails for any reason. If sending lower amount, always send more material than the sample requirement to ensure enough material for QC and prep.

Panel- and whole exome sequencing (PAxKTTRxxx, EXxKTTRxxx):

  • Recommended amount: 500 ng
  • Recommended concentration: > 14 ng/ul
  • Sample requirement: 250 ng
  • Minimum amount: 25 ng (for cfDNA down to 10 ng). Note that larger sample amounts often gives better performance.

If the total amount of DNA is < 250 ng, all available material will be used for one library preparation. A higher input amount increases the likelihood of generating a library of high quality and complexity.
This means that a second preparation is not possible, if the first preparation would fail for any reason. If you would like us to NOT use the whole amount of DNA, contact us when placing the order.

For cfDNA, please send all available DNA. For questions contact support@clinicalgenomics.se.

Sample source: Blood, tissue, saliva, cell free DNA. Due of bacterial contamination, Clinical Genomics do not guarantee successful sequencing of saliva samples.

Container:

  • 1 - 3 samples: 96 well plate or 0.5 - 2 mL tubes
  • > 4 samples: 96 well plate

Information about the 96 well plates:

  • Use Eppendorf twin.tec 96 PCR plates, full-skirted (Eppendorf, cat#: 951020427 or cat#: 0030 128.656)
  • Load the samples into the plate column wise and not row wise. Sample 1 should be in well A1, sample 2 in well B1 etc. Note! Do not leave any empty wells in between samples.
  • If sending more than one plate, only the last plate can contain less than 96 samples.
  • Seal the plate tightly to avoid contamination between wells, with for example Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher AB-0626).

Information about the tubes:

  • 0.5 - 2 mL tubes (for example Eppendorf)
  • Tubes must have flip caps or screw caps with external thread (mark tube and cap with sample id).

Sample buffer:

  • The DNA sample should be eluted in nuclease- free water or Tris-HCl pH 8-8.5.
  • The buffer must not contain any enzyme inhibitors such as EDTA or Sodium azide. TE- or AE buffer cannot be used. Enzyme inhibiting components have a negative effect on the library complexity.

Bait sets:

When ordering whole exome sequencing (EXxKTTRxxx), a bait set should not be specified in the order, since for all EXxKTTRxxx-applications Twist Comprehensive Exome Panel (which covers ca 37 Mb of human protein coding regions) is used.

When ordering panel sequencing (PAxKTTRxxx) bait set has to be specified in the order, with one of the options below.
Contact Clinical Genomics if you wish to add a new bait set or want to have more information about available bait sets.

  • GMCKsolid
  • GMSmyeloid
  • GMSlymphoid
  • GMSsolid

Whole Genome Sequencing of human DNA (PCR-free protocol)

Amount:

Important! If the sample requirements are not followed (with too low concentration and/or volume), the consequence could be less complex libraries and negatively affected data quality.

DNA concentration needs to be measured with fluorometric measurements e.g. Qubit (Invitrogen), not with absorbance measurements.

Always submit > 20 ul. However, ensure that the total amount of DNA is sufficient (see below). For tumour samples more DNA than the "Minimum amount" is used (up to 2000 ng for standard prep or 600 ng for low-input prep), if that amount is available.

Note! If possible, provide the recommended amount which allows for two library preparations in case the first prep fails for any reason. If sending lower amount, always send more material than the sample requirement to ensure enough material for QC and prep.

DNA PCR-free (WGxPCFCxxx):

  • Recommended amount: 2200 ng
  • Recommended concentration: > 25 ng/ul
  • Sample requirement: 1100 ng

Low-input DNA PCR-free (WGxLIFCxxx), costs additionally 1600 SEK/sample compared to standard protocol:

  • Recommended amount: 600 ng
  • Recommended concentration: > 8 ng/ul
  • Sample requirement: 200 ng

Sample source: Blood, tissue (not FFPE), saliva, cell free DNA. Due of bacterial contamination, Clinical Genomics do not guarantee successful sequencing of saliva samples.

Container:

  • 1 - 3 samples: 96 well plate or 0.5 - 2 mL tubes
  • > 4 samples: 96 well plate

Information about the 96 well plates:

  • Use Eppendorf twin.tec 96 PCR plates, full-skirted (Eppendorf, cat#: 951020427 or cat#: 0030 128.656)
  • Load the samples into the plate column wise and not row wise. Sample 1 should be in well A1, sample 2 in well B1 etc. Note! Do not leave any empty wells in between samples.
  • If sending more than one plate, only the last plate can contain less than 96 samples.
  • Seal the plate tightly to avoid contamination between wells, with for example Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher AB-0626).

Information about the tubes:

  • 0.5 - 2 mL tubes (for example Eppendorf)
  • Tubes must have flip caps or screw caps with external thread (mark tube and cap with sample id).

Metagenomic DNA sequencing (PCR-free protocol)

Amount:

Important! If the sample requirements are not followed (with too low concentration and/or volume), the consequence could be less complex libraries and negatively affected data quality.

DNA concentration needs to be measured with fluorometric measurements e.g. Qubit (Invitrogen), not with absorbance measurements.

Always submit > 20 ul. However, ensure that the total amount of DNA is sufficient (see below).

Note! If possible, provide the recommended amount which allows for two library preparations in case the first prep fails for any reason. If sending lower amount, always send more material than the sample requirement to ensure enough material for QC and prep.

DNA PCR-free (WGxPCFCxxx):

  • Recommended amount: 2200 ng
  • Recommended concentration: > 25 ng/ul
  • Sample requirement: 1100 ng

Low-input DNA PCR-free (WGxLIFCxxx), costs additionally 1600 SEK/sample compared to standard protocol:

  • Recommended amount: 600 ng
  • Recommended concentration: > 8 ng/ul
  • Sample requirement: 200 ng

Container:

  • 1 - 3 samples: 96 well plate or 0.5 - 2 mL tubes
  • > 4 samples: 96 well plate

Information about the 96 well plates:

  • Use Eppendorf twin.tec 96 PCR plates, full-skirted (Eppendorf, cat#: 951020427 or cat#: 0030 128.656)
  • Load the samples into the plate column wise and not row wise. Sample 1 should be in well A1, sample 2 in well B1 etc. Note! Do not leave any empty wells in between samples.
  • If sending more than one plate, only the last plate can contain less than 96 samples.
  • Seal the plate tightly to avoid contamination between wells, with for example Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher AB-0626).

Information about the tubes:

  • 0.5 - 2 mL tubes (for example Eppendorf)
  • Tubes must have flip caps or screw caps with external thread (mark tube and cap with sample id).

Whole genome sequencing of microbial DNA (PCR-based protocol)

Important! If the sample requirements are not followed (with too low concentration and/or volume), the consequence could be less complex libraries and negatively affected data quality.

For application MWXNXTR003, analysis of samples will be done once (i.e. no attempt to analyze drop out samples, or samples that do not achieve target of 50x coverage). All submitted samples will be invoiced.
Negative controls should be named NTC# (where #=1,2,3 etc.).

Please contact Clinical Genomics before the order if the samples have been extracted with MagNA Pure 96 or Maelstrom extraction systems.

Note: In order to benefit from the development work we have done on reducing the cost of microbial whole genome sequencing, we request that all projects submitted for this analysis fully adhere to the instructions provided below. Lack of compliance may lead to Clinical Genomics being unable to accept the samples for sequencing, or alternatively refer the samples to a different microbial whole genome sequencing workflow. The alternative workflows are more expensive.

Amount:

DNA concentration needs to be measured with fluorometric measurements e.g. Qubit (Invitrogen), not with absorbance measurements.

Always submit > 15 ul. However, ensure that the total amount of DNA is sufficient (see below).

  • Nextera DNA Sample Preparation Kit (MWxNXTR003):
    • Sample requirement: 50 ng
    • Concentration: All samples must be normalized to 5- 15 ng/ul

Container:

  • 1 - 3 samples: 96 well plate or 0.5 - 2 mL tubes
  • > 4 samples: 96 well plate

Information about the 96 well plates:

  • Use Eppendorf twin.tec 96 PCR plates, full-skirted (Eppendorf, cat#: 951020427 or cat#: 0030 128.656)
  • Load the samples into the plate column wise and not row wise. Sample 1 should be in well A1, sample 2 in well B1 etc. Note! Leave column 12, A12-H12, empty. This column is used for internal controls.
  • If sending more than one whole plate, fill all plates with 88 samples. Only the last plate can have less than 88 samples.
  • Do not leave any empty wells between two samples
  • Seal the plate tightly to avoid contamination between wells, with for example Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher AB-0626).

Information about the tubes:

  • 0.5 - 2 mL tubes (for example Eppendorf)
  • Tubes must have flip caps or screw caps with external thread (mark tube and cap with sample id).

Extraction:

  • We recommend that each plate should include a negative extraction control (using water/EB-buffer instead of sample).
  • Note! We do not analyze samples extracted with MagNaPure 96 (Roche). Please contact Clinical Genomics prior to submission if your samples already are extracted with this system.

Sample Buffer:

  • Extracted DNA cannot be eluted in buffers containing enzyme inhibiting components such as EDTA or Natriumazid. TE- or AE buffer cannot be used. Enzyme inhibiting components have a negative effect on the library complexity.
  • Examples of buffers that are accetable to use are nuclease-free water or buffer EB.
  • Please contact Clinical Genomics prior to submission if your samples already are eluted in non accepted buffers.

Amplicon sequencing (PCR-based protocol)

Important! If the sample requirements are not followed (with too low concentration and/or volume), the consequence could be less complex libraries and negatively affected data quality.

The amplicons need to have a size > 150 bp.

Amount:

DNA concentration needs to be measured with fluorometric measurements e.g. Qubit (Invitrogen), not with absorbance measurements.

  • DNA tagmentation protocol (VWGPDTR001):
    • Minimum amount: 100 ng
    • Volume: > 25 ul

Container:

  • 1 - 3 samples: 96 well plate or 0.5 - 2 mL tubes
  • > 4 samples: 96 well plate

Information about the 96 well plates:

  • Use Eppendorf twin.tec 96 PCR plates, full-skirted (Eppendorf, cat#: 951020427 or cat#: 0030 128.656)
  • Load the samples into the plate column wise and not row wise. Sample 1 should be in well A1, sample 2 in well B1 etc. Note! Do not leave any empty wells in between samples.
  • If sending more than one plate, only the last plate can contain less than 96 samples.
  • Seal the plate tightly to avoid contamination between wells, with for example Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher AB-0626).

Information about the tubes:

  • 0.5 - 2 mL tubes (for example Eppendorf)
  • Tubes must have flip caps or screw caps with external thread (mark tube and cap with sample id).

Sample Buffer:

  • Extracted DNA cannot be eluted in buffers containing enzyme inhibiting components such as EDTA or Natriumazid. TE- or AE buffer cannot be used. Enzyme inhibiting components have a negative effect on the library complexity.
  • Examples of buffers that are accetable to use are nuclease-free water or buffer EB.
  • Please contact Clinical Genomics prior to submission if your samples already are eluted in non accepted buffers.

Transcriptome sequencing (RNA-seq)

Important! If the sample requirements are not followed (with too low concentration and/or volume), the consequence could be less complex libraries and negatively affected data quality.

RNA-samples are easily degraded, therefore it is important to minimize the number of freeze-thawing and to deliver them frozen, preferably on dry ice.

The RNA samples can be handed in to SciLifeLab reception every working day latest at 9.00 or by courier (more info here).

Amount and quality:

DNA concentration needs to be measured with fluorometric measurements e.g. Qubit (Invitrogen), not with absorbance measurements.

Always submit > 20 ul. However, ensure that the total amount of DNA is sufficient (see below).

Note! If possible, provide the recommended amount which allows for two library preparations in case the first prep fails for any reason. If sending lower amount, always send more material than the sample requirement to ensure enough material for QC and prep.

  • Stranded mRNA (RNxPOARxxx):
    • Recommended amount: 600 ng
    • Recommended concentration: > 12 ng/ul
    • Recommended RNA integrity number (RIN-value): > 7
    • Sample requirement: 300 ng
    • Minimum amount: 100 ng. Note that larger sample amounts often gives better performance.

Container:

  • 1 - 3 samples: 96 well plate or 0.5 - 2 mL tubes
  • > 4 samples: 96 well plate

Information about the 96 well plates:

  • Send maximum 30 samples per plate
  • Use Eppendorf twin.tec 96 PCR plates, full-skirted (Eppendorf, cat#: 951020427 or cat#: 0030 128.656)
  • Load the samples into the plate column wise and not row wise. Sample 1 should be in well A1, sample 2 in well B1 etc. Note! Do not leave any empty wells in between samples.
  • Seal the plate tightly to avoid contamination between wells, with for example Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher AB-0626).

Information about the tubes:

  • 0.5 - 2 mL tubes (for example Eppendorf)
  • Tubes must have flip caps or screw caps with external thread (mark tube and cap with sample id).

Sequencing of Ready-Made-Libraries (RMLs)

DNA concentration needs to be measured with fluorometric measurements e.g. Qubit (Invitrogen), not with absorbance measurements.

Fragment size (average size in basepairs) for the libraries needs to be determined and the electropherogram attached to the ticket.

Always submit >20 ul with a concentration of 5 nM or more.

  • Application tag to choose if ordering a part of a flowcell: RMLP15Rxxx
  • Application tag to choose if ordering a whole flowcell: RMLPxxRxxx

Please contact us through support@clinicalgenomics.se if you have any questions.

Container:

  • 1 - 3 samples: 96 well plate or 0.5 - 2 mL tubes
  • > 4 samples: 96 well plate

Information about the 96 well plates:

  • Use Eppendorf twin.tec 96 PCR plates, full-skirted (Eppendorf, cat#: 951020427 or cat#: 0030 128.656)
  • Load the samples into the plate column wise and not row wise. Sample 1 should be in well A1, sample 2 in well B1 etc. Note! Do not leave any empty wells in between samples.
  • Seal the plate tightly to avoid contamination between wells, with for example Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher AB-0626).

Information about the tubes:

  • 0.5 - 2 mL tubes (for example Eppendorf)
  • Tubes must have flip caps or screw caps with external thread (mark tube and cap with sample id).

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