Preventing Contamination Expert Video Tip

Hello Illumina community. I'm Imani Bethel, a sensation applications scientist, and I'm Tim Gilmartin, also a field applications scientist. Many next-generation sequencing applications require some level of DNA amplification, most commonly by polymerase chain reaction or PCR. PCR amplification creates an opportunity for

contamination

is one of the most problematic sources of error in biological experimentation. Contamination can arise simply by transferring a small amount from one sample to another or can be traced back to previously amplified material passed on to the next batch of samples to be amplified in either case. Contamination can have a major impact on project data if the samples used in next generation sequencing library preparation are contaminated, this can cause a drastic increase in background noise in the data and even lead to false discoveries.

Preventing

contamination

is vital to generating reproducible and impactful data. and today we will examine best practices for minimizing the potential for contamination in your laboratory. Preventing contamination ideally starts with lab design if you are performing PCR amplification. Establish two separate areas (one of them used for sample handling before PCR processing) is known as pre-PCR. The two pre-PCR area used for sample handling after processing of a PCR is called post-PCR, If possible use two separate rooms for pre- and post-PCR areas, ensuring physical separation of post-amplification material from pre-amplification samples, the use of two separate rooms also allows for other recommended preventative measures, such as maintaining a pressure of positive air in the pre-PCR area and adding air locks from the pre- to post-PCR area, these steps can greatly reduce the opportunity for previously amplified material to re-enter the pre-PCR area If using two separate rooms are not an option in your facility.

Separate benches or areas are acceptable if separate equipment is used for PCR pre- and post-processing and different personal protective equipment or PPE is used in both areas. This would include items such as gloves, lab coats, eye protection, disposable. shoe and boot covers, strict cleaning schedules must also be maintained, all of which we will discuss in more detail shortly, basically the separate area should be treated as if they were separate rooms. Illumina sequencing in library preparations often requires benchtop centrifuges, vortex bugs, pipettes - Brax Thermal blocks and many other laboratory equipment should not be shared between pre- and post-PCR areas; instead, use separate dedicated equipment in the respective pre- and post-PCR areas, including refrigerators and freezers.

One of the best ways to prevent contamination during library preparation is to process only in one direction from pre-PCR to post-PCR, this unidirectional workflow should prevent potential sources of contamination from returning to the processing area. pre-PCR. These potential sources include tube holders, pipettes, pipette tips, and other laboratory equipment that may contain amplified templates, as well as laboratory gloves. gowns or other PPE that has been used in the post-PCR area be sure to put on new gloves when entering the pre-PCR area and change gloves frequently keep dedicated sets of PPE for the pre- and post-PCR areas store these separate sets of PPE and clean them regularly.

Cleaning prevents contamination from remaining on the PPE and getting into other places. Ideally, a laboratory member who has worked in post-PCR should not return to pre-PCR on the same day if a laboratory member must return to pre-PCR after working in post-PCR carefully put on new PPE. and thoroughly clean all areas in the pre-PCR after your return. Additionally, use dedicated dressing areas near the pre and post PC entrance and exits. These are areas where PPE can be applied or removed before entering or exiting any of the areas. SART an aliquot of all reagents shared between pre-PCR and post-PCR in the pre-PCR area carry the post-PCR agents to the post-PCR area after being sorted into the pre-PCR area;

It's often the simplest details that have the biggest impact and that's definitely the case with the tubing header. To avoid potential contamination while pipetting make sure the tip is seated firmly on the pipettor. Hold the tube head vertically and go slowly. Make sure you have aspirated the correct volume Check the outside of the tip for any unexpected drops of liquid and hold the tube head between 20 and 45 degree angle to achieve optimal flow of liquid out of the tip and avoid splashing here are An online

video

to learn more about proper pipetting, even if you are using perfect pipetting technique, sample-to-sample contamination can still occur due to aerosols, an efficient way to combat aerosol contamination is to use tips pipette or barrier tips resistant to aerosols and liquids.

These types of tips typically use a self-sealing filter tip to prevent liquids or aerosols from coming into contact with the tubing. It is best to avoid spills or splashes of samples by carefully opening or closing swing-down tubes or plates. tubes or plates containing samples to ensure that all sample volumes are collected at the bottom of the well when working with Illumina library preparation kits. Be sure to change tips between each sample when adding or transferring samples or reagent master mixes. change tips between each row and column when adding index adapters remove unused index adapter tools from work area open only one index adapter to avoid losing placement of caps use a fresh new cap to return To seal the index adapter tubes follow the best practices listed in the Illumina reference guide for your The application of a scheduled routine cleaning with 10% bleach or approximately 0.5% sodium hypochlorite is strongly recommended to avoid pollution.

This typically occurs daily and weekly, with thorough cleaning performed weekly and also cleaning your work area before beginning a procedure can proactively remove any contamination. in the area areas to target include pipettes: Brax work tables and other surfaces used control panels for thermal block centrifuges or vortex bugs handles for freezers refrigerators ovens or frequently used drawers or cabinets mice and computer keyboards any areas that commonly touched during normal laboratory operations Often referred to as hot spot solutions, solutions with low concentrations of bleach can lose potency quickly and variably depending on your specific laboratory environment. For best results, prepare a fresh bleach solution daily from a higher concentration solution;

Also, if the surface is bleach resistant, wipe it and then allow 10 to 15 minutes before using a paper towel moistened with water or 70% ethanol to remove residual chlorine, it is highly recommended to include a non-stencil control or NTC in experiments using amplification by including a sample as a non-template control, we can identify contamination. in sample reagents or in the laboratory environment, if the NTC generates amplified products, in addition, monitoring the frequency of contamination can also help identify the root cause of contamination or prevent an increase in the rate of occurrence . Here is a table of DNA sequencing applications available from Illumina. website there are over 100 methods posted on the website with similar quantities available for RNA look for most of these applications all share a common characteristic require some PCR amplification using the best practices we have highlighted in this

video

can help you avoid contamination in your lab, to ensure greater processing efficiency and robust data, be sure to configure your lab to use separate one-way flow and PPE lab equipment.

Focus on good laboratory technique when using pipettes, sample handling, and index adapters. Clean areas frequently and monitor performance with NTC tracking. These tips will help you prevent contamination in your laboratory and the headaches that come with it. Happy sequencing and, as always, thanks for being part of the Illumina community.