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 the prep fails, we will do a second attempt to prep the sample (granted that there is enough material). If the second attempt also fails, we will still invoice the sample. 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

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.

Amount:

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

  • 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.

  • Panel- and whole exome sequencing (PANKTTRxxx, EXOKTTRxxx):

    • Recommended amount: 500 ng (allows for the possibility of two library preps)
    • Required amount: 250 ng (always provide some extra material for QC)
    • Minimum amount: 25 ng (for cfDNA down to 10 ng). Note that larger sample amounts often give better performance.
    • Required concentration: 14 - 200 ng/ul
    • Required volume: 20 - 130 ul (ensure that the total amount of DNA is sufficient)

Sample source:

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

Container:

  • We only accept the following 96 well plates:
    • Eppendorf twin.tec 96 PCR plates, full-skirted (Eppendorf, cat#: 951020427 or cat#: 0030 128.656)
    • Biorad Hard-Shell 96-Well PCR Plates, skirted (Biorad cat#: HSP9601)
  • Place the samples column wise starting in A1 and do not leave any empty wells between samples.
  • If sending more than one plate, only the last plate can contain less than 96 samples.
  • For sealing the plate we recommend using aluminum foil from Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher, cat#: AB-0626).
  • Seal the plate tightly to avoid contamination between wells, for example by using a seal applicator (VWR, cat#: 732-2705).

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

When ordering panel sequencing (PANKTTRxxx) 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)

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.

Amount:

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

  • The DNA needs to be of high quality. If you are unsure about the sample quality, perform a quality analysis (e.g. Tapestation or BioAnalyzer) prior to submitting samples and contact Clinical Genomics for consultation.

  • 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.

  • DNA PCR-free (WGSPCFCxxx):

    • Recommended amount: 2200 ng (allows for the possibility of two library preps)
    • Required amount: 1100 ng (always provide some extra material for QC)
    • Required concentration: 25 - 400 ng/ul
    • Required volume: > 20 ul (ensure that the total amount of DNA is sufficient)

  • Low-input DNA PCR-free (WGSLIFCxxx), slightly higher cost compared to standard protocol:

    • Recommended amount: 600 ng (allows for the possibility of two library preps)
    • Required amount: 200 ng (always provide some extra material for QC)
    • Required concentration: 8 - 400 ng/ul
    • Required volume: > 20 ul (ensure that the total amount of DNA is sufficient)

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:

  • We only accept the following 96 well plates:
    • Eppendorf twin.tec 96 PCR plates, full-skirted (Eppendorf, cat#: 951020427 or cat#: 0030 128.656)
    • Biorad Hard-Shell 96-Well PCR Plates, skirted (Biorad cat#: HSP9601)
  • Place the samples column wise starting in A1 and do not leave any empty wells between samples.
  • If sending more than one plate, only the last plate can contain less than 96 samples.
  • For sealing the plate we recommend using aluminum foil from Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher, cat#: AB-0626).
  • Seal the plate tightly to avoid contamination between wells, for example by using a seal applicator (VWR, cat#: 732-2705).

Metagenomic DNA sequencing (PCR-free 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.

Amount:

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

  • DNA PCR-free (METPCFCxxx):

    • Recommended amount: 2200 ng (allows for the possibility of two library preps)
    • Required amount: 1100 ng (always provide some extra material for QC)
    • Required concentration: 25 - 400 ng/ul
    • Required volume: > 20 ul (ensure that the total amount of DNA is sufficient)

  • Low-input DNA PCR-free (METLIFCxxx), slightly higher cost compared to standard protocol:

    • Recommended amount: 600 ng (allows for the possibility of two library preps)
    • Required amount: 200 ng (always provide some extra material for QC)
    • Required concentration: 8 - 400 ng/ul
    • Required volume: > 20 ul (ensure that the total amount of DNA is sufficient)

Container:

  • We only accept the following 96 well plates:
    • Eppendorf twin.tec 96 PCR plates, full-skirted (Eppendorf, cat#: 951020427 or cat#: 0030 128.656)
    • Biorad Hard-Shell 96-Well PCR Plates, skirted (Biorad cat#: HSP9601)
  • Place the samples column wise starting in A1 and do not leave any empty wells between samples.
  • If sending more than one plate, only the last plate can contain less than 96 samples.
  • For sealing the plate we recommend using aluminum foil from Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher, cat#: AB-0626).
  • Seal the plate tightly to avoid contamination between wells, for example by using a seal applicator (VWR, cat#: 732-2705).

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.

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.

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.).

Amount:

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

  • Nextera DNA Sample Preparation Kit (MWxNXTR003):

    • Required amount: 50 ng
    • Required concentration: All samples must be normalized to 5- 15 ng/ul
    • Required volume: > 15 ul (ensure that the total amount of DNA is sufficient)

Container:

  • We only accept the following 96 well plates:
    • Eppendorf twin.tec 96 PCR plates, full-skirted (Eppendorf, cat#: 951020427 or cat#: 0030 128.656)
    • Biorad Hard-Shell 96-Well PCR Plates, skirted (Biorad cat#: HSP9601)
  • Place the samples column wise starting in A1 and do not leave any empty wells between samples.
  • 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.
  • For sealing the plate we recommend using aluminum foil from Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher, cat#: AB-0626).
  • Seal the plate tightly to avoid contamination between wells, for example by using a seal applicator (VWR, cat#: 732-2705).

Extraction:

  • We recommend that each plate should include a negative extraction control (using water/Tris-HCl 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 Tris-HCl (e.g. EB buffer).
  • Please contact Clinical Genomics prior to submission if your samples already are eluted in non accepted buffers.

SARS-Cov-2 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
    • Required volume: > 25 ul

Container:

  • We only accept the following 96 well plates:
    • Eppendorf twin.tec 96 PCR plates, full-skirted (Eppendorf, cat#: 951020427 or cat#: 0030 128.656)
    • Biorad Hard-Shell 96-Well PCR Plates, skirted (Biorad cat#: HSP9601)
  • Place the samples column wise starting in A1 and do not leave any empty wells between samples.
  • If sending more than one plate, only the last plate can contain less than 96 samples.
  • For sealing the plate we recommend using aluminum foil from Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher, cat#: AB-0626).
  • Seal the plate tightly to avoid contamination between wells, for example by using a seal applicator (VWR, cat#: 732-2705).

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 Tris-HCl (e.g. EB buffer).
  • 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 08.30 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.

  • Stranded mRNA (RNAPOARxxx):

    • Recommended amount: 600 ng (allows for the possibility of two library preps)
    • Required amount: 300 ng (always provide some extra material for QC)
    • Minimum amount: 100 ng. Note that larger sample amounts often gives better performance.
    • Required concentration: > 12 ng/ul
    • Required volume: > 20 ul (ensure that the total amount of DNA is sufficient)
    • Recommended RNA integrity number (RIN-value): > 7

Sample source:

Protocol not compatibel with RNA from FFPE tissue.

Container:

  • We only accept the following 96 well plates:
    • Eppendorf twin.tec 96 PCR plates, full-skirted (Eppendorf, cat#: 951020427 or cat#: 0030 128.656)
    • Biorad Hard-Shell 96-Well PCR Plates, skirted (Biorad cat#: HSP9601)
  • Place the samples column wise starting in A1 and do not leave any empty wells between samples.
  • If sending more than one plate, only the last plate can contain less than 96 samples.
  • For sealing the plate we recommend using aluminum foil from Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher, cat#: AB-0626).
  • Seal the plate tightly to avoid contamination between wells, for example by using a seal applicator (VWR, cat#: 732-2705).

Sequencing of Ready-Made-Libraries (RMLs)

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.

Amount and quality:

  • 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.

  • RML Sequencing (RMLPxxRxxx):

    • Required concentration: > 5 nM
    • Required volume: > 20 ul (ensure that the total amount of DNA is sufficient)

Container:

  • We only accept the following 96 well plates:
    • Eppendorf twin.tec 96 PCR plates, full-skirted (Eppendorf, cat#: 951020427 or cat#: 0030 128.656)
    • Biorad Hard-Shell 96-Well PCR Plates, skirted (Biorad cat#: HSP9601)
  • Place the samples column wise starting in A1 and do not leave any empty wells between samples.
  • If sending more than one plate, only the last plate can contain less than 96 samples.
  • For sealing the plate we recommend using aluminum foil from Thermo Scientific™ Adhesive PCR Foil Plate Seal (Fisher, cat#: AB-0626).
  • Seal the plate tightly to avoid contamination between wells, for example by using a seal applicator (VWR, cat#: 732-2705).

Senast uppdaterad av webmaster

Last Updated