What is the concentration of the NEXTflex DNA Barcodes?
The concentration of the NEXTflex DNA Barcodes is 25 µM.
At what concentration are the NEXTflex PCR primers supplied?
The NEXTflex PCR Primers concentration is 12.5 µM.
Do the NEXTflex DNA-Seq Barcodes Kits contain PCR primers?
Yes, these barcodes are supplied with PCR primers.
Can I order more NEXTflex PCR primers alone?
Yes, the NEXTflex PCR Primers are available in sets of 48, 96, 192, 384 or 768 reactions. Contact [email protected] for more information.
How is the quality of the NEXTflex DNA Barcodes ensured?
Each lot of the NEXTflex barcodes is sequence verified on an Illumina MiSeq.
What type of purification is used when the NEXTflex DNA Barcodes are synthesized?
In order to ensure the highest quality sequencing data, the NEXTflex Barcodes are HPLC-purified, minimizing the risk of mispriming events due to truncations and other data quality-compromising errors.
Preventing Cross Contamination of Samples and Adapter Barcodes During NGS Library Preparation
With the ever-increasing throughput of sequencing platforms, pooling multiple samples at once has become the common method for making sequencing more economical. Because samples are routinely prepared in parallel, great care must be taken throughout the entire library preparation and sequencing process to prevent cross contamination of the samples, or the unique barcodes that are used to identify each individual sample. Even very low amounts of cross contamination can be devastating to a sequencing experiment, costing thousands of dollars and countless hours of work. The following will identify areas of possible contamination and suggests methods of prevention.
Cross Contamination of Library Template
Cross contamination of starting material is just as damaging as, and is often mistakenly identified as barcode cross contamination. When multiple samples are prepared at the same time or multiple libraries are produced in parallel, there is a risk that individual samples may contaminate each other. The best way to avoid this type of contamination is to perform all sample and library preparations individually, thus eliminating all risk of cross contamination. If doing all sample and library preps individually is not a feasible solution, then strict measures need to be employed to keep your samples pure.
Good practices include:
- Regularly clean pipettes and work surfaces.
- Use aerosol barrier pipette tips.
- Change pipette tips between each and every step
- Use pierceable seals on plates during all pipetting steps. We use Sigma’s X-Pierce Seals.
- Leave an empty well between each sample in the plate when possible.
- Use a plate centrifuge to spin down plates before use.
- Mix samples by pipetting up and down with a pierceable seal in place.
- Never mix plates on vortexers. Placing a plate on a vortexer to mix samples or barcodes has been proven to lead to cross contamination, even if the plate appears to be securely sealed. We recommend that the NEXTflex-96 Barcodes be mixed by pipette and never on a vortexer.
Determining Cause of Contamination
In cases where sample contamination in amplicon-seq libraries is suspected because samples appear to be present in multiple barcoded libraries after demultiplexing, you need to determiner whether you cross-contaminated your starting material or the set of barcodes adapters. The cause can be determined by running a Bioanalyzer trace on the suspect library. If the contamination was caused by barcode impurity, the library in question should have only one amplicon peak, as distinct barcodes would not be distinguishable on a Bioanalyzer. Alternatively, if the issue is due to cross-contamination of starting material, the same barcode should be ligated to multiple amplicons and should be evidenced by multiple peaks on the Bioanalyzer. An example can be seen in Figure 1. Amplicon-seq libraries with sample contamination, along with non-contaminated samples, were run on a Bioanalyzer (Figure 1). The pure samples contained only one amplicon (Figure 1A); however, the contaminated samples contained multiple amplicons (Figure 1B). Running the starting material on the Bioanalyzer and observing multiple peaks in the contaminated samples further confirmed this result. This simple Bioanalyzer test clearly indicates that the contamination issue was a starting material problem and not a barcode problem.
Since most sequencing applications are not pure amplicon samples but complex mixtures in which slight contamination might go unnoticed, amplicon-seq datasets are uniquely able to demonstrate how to prevent cross-contamination.
Preventing Barcode Contamination during Library Preparation
As with sample preparation, using aerosol barrier tips, changing tips after every use, mixing by pipetting, and using pierceable seals during any pipetting steps, are all ways to reduce contamination during library preparation. All of these precautions are important not only during the enzymatic steps, but during the cleanup steps as well.
Other important methods to employ if using plated barcoded adapters are:
- Making sure that the plate seal is securely affixed to all of the individual wells to prevent any aerosol contamination during incubation and cycling. A good way of accomplishing this is to run your finger down all of the columns and across all of the rows after you put the seal on the plate or use an adhesive film applicator.
- Immediately spinning down plates upon sealing and after removal from the thermal cycler.
- Gently removing plate seals by peeling them away from the wells in order to prevent splashing across wells.
- Applying pierceable seals during any pipetting steps.
Handling of Barcoded Adapters
When using barcoded adapters for multiplexing, it is crucial that they be added in a systematic manner that ensures only the correct barcode is added to each sample. We offer our barcodes in both tubes and in plates, and there are different ways to handle both formats.
Methods to accomplish the accurate addition of barcodes are:
- Only open one tube at a time when using barcodes that are provided in tubes.
- Spin down tubes after thawing/mixing.
- Organize rows of pipette tips so that each tip corresponds to a particular adapter. Doing this will decrease the chance of unintentionally adding two separate barcodes to the same sample.
- When using barcodes that are provided in plates, make sure that the plate has been spun down before using. Our NEXTflex-96 barcode plate is sealed with a pierceable heat seal that does not need to be removed. This greatly reduces the chances of contamination occurring during the removal of seals.
- After the barcodes have been added, simply place another seal on top of the pierced seal to store the remaining volume.
Sequencing Platform Issues
Even after producing individually barcoded libraries that are contamination free, problems can still arise during the sequencing run itself. The most troubling issue that has come to light is that MiSeq users have noticed small amounts of sample carryover between sequencing runs. Some good discussions of this issue can be found on SEQanswers. It appears that there can be up to 0.5% of the previous sample left over after cleaning the machine between runs, which will then be detected during subsequent runs. This is especially problematic in clinical studies and any other application attempting to uncover low prevalence mutations.
Illumina says that with prompt and thorough cleaning of instruments, the percentage of reads from carryover is 0.1% or less. Illumina recommends washing only with water and Tween, and discourages users from using bleach or other additives that may damage the machine. Although 0.1% may seem like a trivial amount, it is still far too high for many applications.
One of the easiest fixes suggested on SEQanswers is to alternate the sets of barcodes used between sequencing runs. Rotating three sets would guarantee that the machine is washed at least three times before a barcode was used again. Any template that is carried over from a previous run should not have a barcode that is on the next run’s sample sheet, and should therefore be thrown out during demultiplexing. While this may be a good solution for users that sequence in-house, it does not solve the problem for groups utilizing core facilities, so a further fix from Illumina may still be necessary. For further advice on how to deal with this issue, Genome Biology also has an informative article that discusses data analyses strategies that can be employed.