1. General Considerations

Transcription

1 Rev: F Page: 1 of 11 For successful completion of your project it is essential to adhere to the following instructions. This document will provide guidelines on DNA or RNA sample quality requirements and steps necessary for sample submission to CNAG-CRG. Contents 1. General Considerations Sample Quality and Quantity Requirements Labelling and Packaging Requirements Sample information submission to CNAG External Server Shipping samples to CNAG General Considerations Experimental design of your project must be agreed upon and your project quoted by the Project Manager (Berta Fusté, berta.fuste@cnag.crg.eu or projectmanager@cnag.crg.eu). Questions related to DNA and RNA sample requirements and shipment details should be directed to the CNAG Biorepository (Lídia Agueda, Biorepository Laboratory Manager, lidia.agueda@cnag.crg.eu or Ana González, ana.gonzalez@cnag.crg.eu). Questions related to externally prepared libraries should be directed to the Sample Preparation Laboratory (Julie Blanc, Sample Preparation Manager, julie.blanc@cnag.crg.eu). 2. Sample Quality and Quantity Requirements Sample quality directly affects the sequencing quality and subsequent bioinformatics analysis. Therefore, CNAG has extensive sample quality control procedures to ensure submitted samples conform to requirements for downstream processing. If your samples can not meet CNAG specifications (Table 1), please consult with the CNAG Project Manager before shipping your samples. CNAG will inform the collaborator about the quality control results if any of the samples do not meet CNAG specifications. The collaborator must decide whether: i) to replace failed samples or Lidia Agueda Berta Fusté/Ana Gonzalez Marta Gut 15/02/2017

2 Rev: F Page: 2 of 11 ii) proceed with the failed samples as at risk and will be subjected to billing regardless of data quality. Please consult the Project Manager for further details. The CNAG Biorepository can also provide advice on DNA/RNA extraction methods if the extraction has yet to be performed. Moreover, for already extracted samples, the collaborator can also give feedback regarding the samples quality before samples are shipped. INPUT: The amount/quality of the input material needs to be discussed with the CNAG Project Manager as it may vary in function of experiment type and/or the genome size of the studied organism. Most commonly used protocols and sample amount requirements are listed in Table 1. Input material quantity should be determined by fluorescence-based quantification methods such as Qubit or Quant-It. However, when this is not possible and only absorbance based quantification is available always provide as much material as possible. QUALITY: Sample quality requirements are listed in Table 2. Lidia Agueda Berta Fusté/Ana Gonzalez Marta Gut 15/02/2017

3 Table 1. Protocols and sample amount requirements Sequencing protocol MINIMAL quantity requested (fluorescence based quantification method) Concentration range (ng/ul) Regular Whole Genome 5 ug Low Input Whole Genome 400 ng * De Novo Genome Assembly 8,5 ug * Nanopore 3 ug * Whole Genome Bisulphite 5 ug Low Input Whole Genome Bisulphite 1,5 ug CustomCapture - Bisulphite 7 ug OX- Whole Genome Bisulphite 7 ug Regular Exome and Custom Capture (Agilent) 4,5 ug Low Input Exome and Custom Capture (Agilent) 800 ng* Regular Exome and Custom Capture (Nimblegen) 2 ug Low Input Exome and Custom Capture (Nimblegen) 1 ug* Medexome 2 ug / 1 ug* Amplicon Sequencing Clone Sequencing Same protocols as Whole Genome Sequencing Same protocols as Whole Genome Sequencing Mate Pair 10 ug Total RNA Sequencing 2 ug Stranded total RNA or mrna Sequencing 2 ug total RNA or 400 ng depleted RNA for total RNA; for depleted RNA Regular mrna-sequencing 2 ug total RNA or 400 ng depleted RNA for total RNA; for depleted RNA Ultra-Low Input mrna Sequencing * * Hi-C protocol * * Genotyping by Sequencing *Contact Project Manager for further details Pilot Phase, 1 enzyme: 1 sample 4 ug; samples 1 ug Pilot Phase, 2 enzymes: 1 sample 7 ug; samples 2 ug Large Scale: 400 ng 20-50

4 Table 2. Sample quality requirements Sample type Quality requirements gdna Pure DNA, free of RNA contamination. Optical Density measurements: OD 260/ and OD 260/ Depending on the extraction method employed, RNAse treatment is required. High molecular weight DNA, no degradation smear Free of other species DNA contamination Free of PCR inhibitors Sample buffer must be water or 10Mm Tris/1mM EDTA Quantified by fluorescence-based method specific for dsdna; Absorbance based quantification is inadequate (Nanodrop or equivalent) Whole Genome Amplified DNA Contact Project Manager Always refer to amplification method employed in data submission manifest FFPE DNA Contact Project Manager PCR amplicons Contact Project Manager Always provide amplicon size in data submission manifest Cloned DNA Contact Project Manager Always provide insert size in data submission manifest Total RNA Pure RNA, free of DNA contamination Good integrity. Bioanalyzer profiles RIN>8 mrna samples must be free of rrna. By means of Bioanalyzer profiles, rrna contamination <22% Sample buffer must be water Quantified by fluorescence-based method specific for RNA; Absorbance based quantification is inadequate (Nanodrop or equivalent) FFPE RNA Contact Project Manager

5 Page: 5 of Labelling and Packaging Requirements Use only CNAG provided material for sample shipment. CNAG will provide the material once project design is completed by the CNAG Project Manager. Sent material might consist of: i) screw cap tubes and labels with barcodes for individual samples. Tube labels contain project name, material type and unique sample ID. Of note, the correct use of the provided selflaminating labels requires to stick the white part with text and go around the tube with the transparent part covering the barcode and text on the white part for further protection. Text orientation from bottom to top. For individual screw cap tubes, never add extra Parafilm sealing, the tubes have an anti-leakage system in the cap (rubber O-ring). It is important to use a container such as a box with interior racks/holders in order to keep all samples grouped and ensure avoid crushing during shipping. Cotton and absorbent papers can be used to prevent tubes from moving around inside the container. ii) 96-well plates, labels for plates, strip caps and sealing foil. Plate labels contain a unique ID and the project name (one plate ID is linked to up to 96 individual sample barcodes). For plate shipment, we strongly recommend using strip caps and sealing foil for proper sealing as shown in the following images:

6 Page: 6 of 11 Never use your own tubes/plates or non-cnag labels for samples identification. Additional material can be requested to CNAG Biorepository. RNA samples must be shipped in dry ice (frozen) and DNA samples can be shipped with blue ice/cooling blocks during shipment (refrigerated) or at room temperature. 4. Sample information submission to CNAG External Server Once a project / subproject is defined with CNAG Project Management, sample / plates unique IDs will be assigned according to number and type of samples and the tubes / plates will be provided to the collaborator. The CNAG Biorepository will also provide the collaborator with an URL link to the submission site (external server, FOR-002) that allows data collection. Once submitted, FOR-002 provides the entire information for appropriate identification and subsequent analysis of the samples, which will be automatically integrated into the CNAG Laboratory Information Management System (LIMS). The correct completion of this document is crucial for the success of the project. FOR-002 must be completed in English and verification by two persons is highly recommended, as any error at this stage will be detrimental to the project.

7 Page: 7 of 11 Contact CNAG Biorepository for any questions or doubts regarding the FOR-002 completion (lidia.agueda@cnag.crg.eu or ana.gonzalez@cnag.crg.eu). CNAG assigned IDs that appear on the external server can be used in different sample shipment batches. Just select and submit the barcodes used for each shipment. Next time the URL is used it will only display the unused barcodes. External server auto-fill tips: - Hover over field names for more details - To fill a whole column with same value: fill in the 1 st row and Ctrl + space or Ctrl + shift - Copy/paste from excel: copy and go to 1 st row in the column and paste the whole column, ensure number or copied cells and rows match. - No special characters are accepted ( ; &; ) - Species: type species name in Latin and select from the displayed list

8 Page: 8 of 11 CNAG external server (FOR-002) contains the following fields: Field name LAB_CENTER All fields are mandatory except for those labeled Optional. Field description: Laboratory identifier. Optional, useful for projects with several participant centers. COHORT_NAME SAMPLE_BARCODE REPLACEMENT_OF SAMPLE_NAME SAMPLE_ TYPE FIXATIVE SPECIES MATERIAL_SOURCE Cohort identifier. Optional, useful if samples belong to different study groups. Sample unique identifier. Provided by CNAG in the same order as printed labels. Check that barcodes in the file correspond to the shipped barcode labels. For PLATES, sample barcodes are already assigned to a unique plate position. Sample barcode of the original sample that is being replaced by this new sample. Mandatory when sending additional material. When a sample is additional material for a previous one, SAMPLE_NAME must be the exact same (see further details below). Sample unique identifier. No patient name or surname should appear in the field. Two aliquots from same sample must have same SAMPLE_NAME but different SAMPLE_BARCODE. When additional material from the same sample is required, both samples must have same SAMPLE_NAME but different SAMPLE_BARCODE. If there are different samples from the same individual (i.e. normal/tumor; treated/untreated ) those must have different SAMPLE_NAME. When two or more experimental replicates need to be sequenced, they must have different SAMPLE_NAME and different SAMPLE_BARCODE. Use only alphanumerical characters (no spaces, dashes or dots). (See further details below). Type of material (gdna, total RNA, small RNA ). Provided by CNAG. Fixative employed for sample conservation if any. Mandatory for FFPE samples. Species from which the DNA/RNA has been obtained. Use NCBI Taxonomy Browser compatible species names. For non-human samples, add known/approximate genome size in the COMMENTS column. Specify the source (e.g. whole blood, buccal swabs, FFPE tissue, liver, whole organism, etc.) from which the DNA/RNA was obtained.

9 Page: 9 of 11 EXTRACTION_METHOD RESUSPENSION_BUFFER INITIAL_VOLUME STOCK_CONCENTRATION ABSORBANCE_RATIO_ 260/280 ABSORBANCE_RATIO_ 260/230 SEX STATUS PEDIGREE_NUMBER FATHER MOTHER GEOGRAPHIC_ORIGIN PLATE_BARCODE PLATE_POSITION COMMENTS Nucleic Acid extraction method employed. Please specify kit and manufacturer, if known. If samples are whole genome amplified, specify amplification protocol employed. Buffer used in final resuspension for the material extraction. Sample volume provided in µl. Exact volume provided to CNAG, no approximations. Sample concentration in ng/µl. Accepted concentration may vary according to the project characteristics, please, discuss with CNAG staff. If the quantification method employed is not fluorescence-based (Qubit/Picogreen); please specify it in COMMENTS column. ABSORBANCE RATIO 260/280 If samples are quantified by absorbance methods. ABSORBANCE RATIO 260/230 If samples are quantified by absorbance methods. Sex of the individual Unknown/Male/Female/Other Status of the individual unknown or not applicable / unaffected or normal or control or wild type / affected or tumor or index case. Pedigree identifier. Mandatory only for family studies. Members of same family will have same PEDIGREE identifier. See example below. Sample_name or CNAG barcode of the father of this individual. Optional, mandatory for family studies. Sample_name or CNAG barcode of the mother of this individual. Optional, mandatory for family studies. Geographic origin of the sample. Plate unique identifier. Provided by CNAG. Check that barcodes in the file correspond to the shipped plate labels. For PLATES, sample barcodes are already assigned to a unique plate position. Plate position. Provided by CNAG. For PLATES, sample barcodes are already assigned to a unique plate position. Any comments that the collaborator wishes to add, and/or any of the previously mentioned: For non-human samples, add known/approximate genome size. Quantification method (if different than Qubit/Picogreen). Optional.

10 Page: 10 of 11 Example of data entry for pedigrees: Sample_Barcode Pedigree_number Father Mother AA AA AA AA0001 AA0002 AA AA0001 AA0002 AA AA0001 AA0002 Sending replacements or additional material to CNAG: a) if any sample needs to be replaced by a NEW ONE (new individual), use a new barcode for sample identification and fill in FOR-002 with the new sample_name (different from the sample being replaced). It is also recommended to fill in comments column with to sequence instead of sample_barcode of the replaced sample. b) if additional material from the SAME SAMPLE needs to be sent, use a new barcode for sample identification, fill in FOR-002 with exactly the same sample_name and fill in the column replacement_of with the barcode of the sample. Once FOR-002 is completed and BEFORE sample shipment, submit it using SUBMIT button and inform by to CNAG Biorepository (lidia.agueda@cnag.crg.eu/ana.gonzalez@cnag.crg.eu).

11 Page: 11 of Shipping samples to CNAG Check that all samples conform to our quality standards and that they are prepared and packaged according to the guidelines given above. Please make sure to notify CNAG Biorepository staff for every sample batch before shipment. Data submission (FOR-002) MUST be completed BEFORE any sample shipment and date of delivery needs to be confirmed. For non-eu shipment. Additional documentation could be requested by the custom authorities. In order to avoid damage of your samples retained in custom premises it is mandatory to contact CNAG Biorepository staff before any sample shipment. Any importation without appropriate documentation will be rejected and parcel will be returned to the sender. Provide the shipment tracking information whenever possible. This is mandatory for non-eu shipments. Parcel reception times: Send parcels preferably at the beginning of the week. Monday to Thursday 8-16h; Friday 8-12h No reception on Saturday, Sunday and local bank holiday CNAG will not be responsible for parcels received outside of these time frames or without prior notification of parcel shipment. Please, confirm with CNAG Biorepository staff the reception timetables during bank or summer holiday or Christmas period. Shipment address: ATT. Lídia Agueda, PhD / Ana González, PhD Centre Nacional de Anàlisi Genòmica (CNAG-CRG) Parc Científic de Barcelona Torre I C/Baldiri i Reixac, 4 Barcelona Spain