Sample requirements

 

 

General recommendations

(Last edited 15/03/2018; sample requirements v3.15)

We recommend that you send us your quality control data before submitting your samples to the facility, to allow our project management team to advise you on the suitability of your samples for Illumina sequencing. We request that you send your quality control (QC) data for each of your samples, at the latest, when they are submitted to us. We will not continue with your project until we have received these data. If you are having difficulties in obtaining samples of sufficient quality or quantity then we can advise you on how to proceed. If you choose to submit samples that are not of sufficient quality or quantity then it is possible for us to proceed with your project at your own risk, with the understanding that such samples are likely to yield lower quality data.
 

Quantity measurements
 

We recommend that the quantity of your samples be measured using fluorimetric quantification methods such as Qubit Quant-iT or Picogreen for dsDNA and Ribogreen for RNA (or another similar fluorimetric method). Alternatively nucleic acid concentrations can be estimated by gel electrophoresis using a ~0.8% agarose gel loaded with your samples alongside a dilution gradient of DNA or RNA standards that spans concentrations greater and less than that of the estimated sample concentration. Gels should be post-stained as opposed to including the nucleic acid stain in the gel, in order to visualize low molecular weight contaminants as well as high molecular weight DNA. We do not recommend using Nanodrop or other spectrophotometric methods for estimating nucleic acid concentrations, as they are generally unreliable. If your samples have been quantified using spectrophometry-based methods, we strongly recommend that you supply us with twice the recommended amount (see below). Please send samples to us at the required concentration. If your samples are of a significantly higher or lower concentration please dilute or concentrate them and re-quantify them.
 

Quality measurements
 

We recommend that the quality of your samples be determined by measuring A260:280 using a spectrophotometry-based method. The A260:280 will enable the determination of whether your sample contains a high amount of protein contamination, which can interfere with downstream processes. If your samples fail this QC requirement initially, it is often possible to perform further purifications such as a phenol chloroform extraction or column based methods to remove the contaminating material. This step needs to be conducted by you and if the samples you have sent to Edinburgh Genomics fail this QC step, then they will be returned to you at your own expense.
 

Sample requirements
 

All our supported protocols have been tested and validated by our laboratory staff using control samples of known quality and quantity, and for each protocol we have devised a set of quality control thresholds that maximise the chance of generating high quality libraries. For convenience, we provide two sets of thresholds, a recommended level and a minimum level (see figure below). Note that these requirements may differ to those recommended by technology suppliers. 

The recommended level includes an allowance for QC measurements in our facility and enough material remaining after QC to generate two libraries, should the first attempt fail for technical reasons. Providing us with the recommended requirement is the “best case scenario” and we advise you to provide samples that meet our recommended requirements wherever possible. Note that we will not charge you to generate a replacement library in the case of initially failed library preparations if your samples have met our recommended requirements. If you are unable to provide us with samples of the recommended level then you may provide us with samples that are equal to or greater than the minimum level.

The minimum level includes an allowance for QC measurements in our facility and enough material remaining after QC to generate a single library. Note that if library preparation fails, we will not charge you, but you will need to supply more material for a second attempt. Please note that this is the strict minimum level for your samples to pass our QC.

Your samples will fail QC if they do not meet our minimum requirements. In this case we strongly recommend that you provide us with replacement samples. If the samples cannot be replaced, you may decide to proceed at your own risk. Note that in this case, we will charge you for library preparation regardless of the outcome. 
 

Sample Submission
 

When you are ready to submit your samples, please send them to the address below:

Edinburgh Genomics
Ashworth Laboratories, Lab G03A
Charlotte Auerbach Road
The King's Buildings
Edinburgh, Scotland
EH9 3FL
 

Specific advice for different samples and technologies:

 

Illumina sequencing (Genome Science)

 

Genomic DNA for TruSeq Nano, RAD, ddRAD library preparation

We require DNA that is free of RNA, minimally degraded, and free of other contaminants.

Please assess the integrity of your gDNA samples by running 2-5μL of each sample on 0.8-1% agarose gel(s) against a 1kb DNA ladder. gDNA should have a high molecular weight band (>12kb) with little/no evidence of degradation (smearing) and minimal RNA contamination (please see Figure 1). Failure to meet either of these criteria can compromise library preparation. Please attach gel pictures to the sample submission form.

To remove RNA from your samples, we recommend that you first treat your samples with a broad spectrum RNase, such as Riboshredder RNase blend (Epicentre Cat # RS12100) post DNA extraction. Residual RNA can be detected as a small band (<200bp) on your agarose gel. If you observe an RNA band that is greater than 10-20% of total nucleic acids, we suggest that you repeat the RNase digestion step. Note that samples must be purified after RNAse treatment. Nextera and TruSeq Nano are particularly sensitive to RNA contamination so please be mindful of this in the preparation of your samples. Please note that we do not currently offer RNase treatments as part of our service.


Figure 1: The marker is a 1Kb ladder (Promega, G5711) with DNA fragments ranging from 250bp to 10Kbp.  The gDNA samples range from very good quality HMW gDNA with very little degradation or RNA contamination (Image 10) to extremely degraded gDNA (Image 1). gDNA samples in images 10 to 7 would pass sample QC.   gDNA samples in images 6 or less would fail sample QC showing greater levels of RNA contamination or DNA degradation.

 

ChIP DNA for ChIP-seq

Please assess the average size (and range) of ChIP enriched DNA running on an Agilent Bioanalyser 2100 DNA 1000 chip (or equivalent) if the expected size is < 1kb. If you do not have access to a Bioanalyser or are dealing with fragments of >1kb, samples can be run on a 1.2-2% agarose gel, against a 100bp ladder. Please attach Bioanalyzer traces or gel pictures to the sample submission form. Note, Illumina recommends a DNA insert size range of 200-400 bp. Please see table below for concentrations and volumes. Please notify us if your ChIP enriched DNA is >1kb in size prior to submitting your samples as we will require additional steps to process it correctly.

PCR products for Nextera XT library preparation

We strongly recommend that you initially optimize the PCR conditions to ensure the amplification of a single product. Assess size (and range) of PCR products by running on an Agilent Bioanalyser 2100 DNA 1000 chip (or equivalent) if the expected size is < 1kb. If you do not have access to a Bioanalyser or are dealing with fragments of >1kb, samples can be run on a 1.2-2% agarose gel against a 100bp ladder. Please attach Bioanalyzer traces or gel pictures to the sample submission form. Please note, samples must NOT be dissolved in a buffer that contains EDTA! We recommend EB (10 mM Tris pH7.5) or nuclease-free dH2O.

Genomic DNA for Nextera, Nextera XT, Nextera Mate-Pair and Nextera Rapid Capture library preparation

Please assess the integrity of your gDNA samples by running 2-5μL of each sample on 0.8-1% agarose gel(s) against a 1kb DNA ladder. gDNA should have a high molecular weight band (>12kb) with little/no evidence of degradation (smearing) and minimal RNA contamination (please see Figure 1). A260:280 values should be 1.8 or greater. High quality DNA is essential for successful Nextera projects as degraded genomic DNA may result in over-fragmentation of DNA to below the desired size range and poor quality libraries.

To remove RNA from your samples, we recommend that you first treat your samples with a broad spectrum RNase, such as Riboshredder RNase blend (Epicentre Cat # RS12100) post DNA extraction. Residual RNA can be detected as a small band (<200bp) on your agarose gel. If you observe an RNA band, we suggest that you repeat the RNase digestion step. After the RNase digestion step, samples should be cleaned up with Zymo gDNA clean and concentrate kit, and eluted with an appropriate amount of buffer. Please note, it is important that your samples are NOT dissolved in a buffer that contains EDTA! We recommend EB (10 mM Tris pH 7.5) or nuclease-free dH2O. 

Library Preparation Protocol Input Material Diluent A260/ 280 Recommended Amount [ng] Recommended Concentration [ng/uL] Minimum Amount [ng] Minimum Concentration [ng/uL] Minimum Volume (uL)
TruSeq DNA PCR-Free 350bp gDNA TE >1.7 2200 37 1200 20 60
TruSeq DNA PCR-Free 550bp gDNA TE >1.7 4400 74 2400 40 60
TruSeq Nano 350bp Gel Free gDNA TE >1.7 250 9 150 5 30
TruSeq Nano 550bp Gel Free gDNA TE >1.7 450 9 250 5 50
TruSeq Nano 350bp Pippin Selected gDNA TE >1.7 250 9 150 5 30
TruSeq Nano 550bp Pippin Selected gDNA TE >1.7 450 9 250 5 50
NEB ChIP seq ChIP DNA EB >1.7 30 1 15 0.5 30
cDNA (TruSeq Nano 350bp Gel Free) cDNA TE >1.7 1100 19 600 10 60
Nextera DNA gDNA  EB >1.8 150 7.5 100 5 20
Nextera DNA XT gDNA  EB >1.8 55 5 55 5 11
Nextera Rapid Capture gDNA  EB >1.8 150 7.5 100 5 20
Single-digest RAD (24-plex) gDNA TE >1.8 580 14 336 8 42
Single-digest RAD (<24-plex) gDNA TE >1.8 1090 28 600 15 40
Single-digest RAD (24-plex; normalised concentration)  gDNA TE >1.8 =600 =10 na na 60
Single-digest RAD (<24-plex; normalised concentration) gDNA TE >1.8 =1200 =20 na na 60
Double-digest RAD (24-plex) gDNA TE >1.8 580 14 336 8 42
Double-digest RAD (<24-plex) gDNA TE >1.8 1090 28 600 15 40
Double-digest RAD (24-plex; normalised concentration)  gDNA TE >1.8 =600 =10 na na 60
Double-digest RAD (<24-plex; normalised concentration) gDNA TE >1.8 =1200 =20 na na 60
Sure Select XT 3ug Method gDNA TE >1.8 6230 45 3243 23 141
Sure Select XT 200ng Method gDNA TE >1.8 450 9 250 5 50
Sure Select XT2 1ug Method gDNA TE >1.8 2200 37 1200 20 60
Sure Select XT2 100ng Method gDNA TE >1.8 250 9 150 5 30

 

Total RNA for TruSeq mRNA-seq, TruSeq stranded mRNA-seq and TruSeq stranded Total RNA

We recommend that all RNA samples be treated with DNase prior to submission. We suggest the use of Turbo DNA-free (Life Technologies Cat # AM1907). Integrity of RNA samples is extremely important, so we suggest that you check this by running all samples on an Agilent Bioanalyser 2100 RNA nano chip, or equivalent. For most vertebrate species a (RNA Integrity Number) RIN value of ≥ 7.0 is acceptable. Please see figure below. The sample on the left is of good quality, the sample in the middle is of moderate but still acceptable quality, and the sample on the right is of poor quality and would fail QC. If you do not have access to a Bioanalyser or similar instrument, samples can be run on a 1.2% agarose gel. The 28S and 18S bands should be 1.5-2.0x brighter than the 18S band. Please attach Bioanalyzer traces or gel pictures to the sample submission form.  These recommendations are for vertebrate species, so please interpret your results in light of the recommendations that are relevant to the species you are working with. If your RNA samples are of an insufficient quantity/concentration to analyse using gel electrophoresis or bioanalyser, please consult with our Project Management team.

Small RNA for RNA-seq

Preparation of RNA for small RNA sequencing requires special attention, as small RNAs will be removed when typical protocols are used for extraction of total RNA. Total RNA is used as input to the Small RNA sequencing library preparation protocol so any kit that preserves the smaller RNA species is suitable for library preparation. These include Trizol and the Qiagen miRNeasy kits. The quality of the RNA required for sequencing library preparation is the same as for standard RNA sequencing library preparation and should be tested in the same way.

Bacterial Total RNA

All bacterial RNA samples should be treated with DNase prior to submission. We suggest the use of Turbo DNA-free (Life Technologies Cat # AM1907). The success of the DNase treatment should be tested using PCR on the RNA for a known bacterial gene. Integrity of RNA samples is extremely important, so we suggest that you check this as indicated for Total RNA above.

 

Library Preparation Protocol Input Material Diluent A260/ 280 Recommended Amount [ng] Recommended Concentration [ng/uL] Minimum Amount [ng] Minimum Concentration [ng/uL] Minimum Volume (uL)
TruSeq mRNA Stranded RNA-seq Total RNA Nuclease Free Water >1.9 2200 37 1200 20 60
TruSeq smRNA Total RNA Nuclease Free Water >1.9 3000 300 2000 200 10
TruSeq Total Stranded RNA-seq Total RNA Nuclease Free Water >1.9 3000 150 2000 100 20
Bact TruSeq Total Stranded RNA-seq Total RNA Nuclease Free Water >1.9 2357 63 1357 36 38

 

User prepared libraries

Please assess the quality of your library by running on the Agilent Bioanalyser 2100 DNA 1000, High Sensitivity chip or equivalent. A good library should be free of any smaller or larger products than the target size for the library preparation technique. When submitting libraries to Edinburgh Genomics please report average size (and range) of the library. If your library has multiple peaks it will be difficult to accurately determine the concentration of DNA in the library for loading onto the sequencer. DNA products in the sequencing library of less than 200 bases (including primer dimer) can be removed using Ampure beads (Beckman Coulter) in a 1:1ratio of sample to beads. Follow the Ampure bead protocol as supplied with the beads at all times. Please note that we do not currently offer library cleanup as one of our services.

The nM concentration of each sample can be calculated using the formula:         

  • nM =  (ng per uL of DNA x 1,000,000) / (Number of base pairs x 660)

If you have difficulty achieving the required concentration and volume, please contact Edinburgh Genomics (edgenomics-admin@ed.ac.uk) to discuss the options available.

If you plan to submit a library requiring custom primers, primer design will be your own responsibility, but you are welcome to contact us ahead of time if you require assistance. When you order your primers, we recommend TruGrade from Integrated DNA technologies (IDT). Primers should be resuspended to a concentration of 100 μM in TE buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0). At the time of library submission, we require that you send us 15 μL of each primer.

Primer information should be submitted to Edinburgh Genomics in the sample submission form, both by email and as a hard copy. We will dispose of your primers at the end of each project, so for any subsequent projects, fresh aliquots of primers should be sent.

Sequencer type Diluent Recommended Concentration Minimum Concentration Minimum Volume (uL)
MiSeq and NovaSeq EB-Tween >5nM** >2.5nM** 15 (1 lane)
MiSeq and NovaSeq Additional Lanes EB-Tween >5nM** >2.5nM** Each Additional lane add 6ul

**Use the following equation for nM = (ng per uL of DNA x 1,000,000) / (Number of base pairs x 660)        
 

Arrays (Genome Science)