General guidelines for good scientific practice for NGS workflows

For NGS workflows, and specifically RNA sequencing, the laboratory environment, equipment and reagents should be kept free of contamination, in order to avoid degradation of the sample(s) by RNases or acquisition of compromising results due to contamination with DNA.

Key Points to Maintaining a Safe and Clean Laboratory Environment and Good Scientific Practice:

• Separate laboratory areas to prevent cross contamination, e.g., culturing and sample collection should be separated from labs where molecular assays are run. For PCR-based and NGS assays, adhere to separation of pre- and post-PCR laboratories to avoid contamination of assay material (e.g., sample, library prep components, or equipment) with PCR products (amplicons). For more information, please see: Is separating Pre-PCR and Post-PCR working areas necessary?

• Follow the workflow and direction of each area to avoid contamination during molecular procedures, e.g., always move your samples from pre-PCR to post-PCR labs, but never in the opposite direction; always change gloves and if necessary, also lab coats and other protective gear.

• Ensure your laboratory environment, equipment and reagents are suited for your experiment, e.g., when working with RNA, ensure the surroundings and materials are free from RNases (see General Guidelines for Lexogen Kit Use for more details).

• Use appropriate controls to check your workflow, experiments and aid with the interpretation of results. Controls ensure the experiment is carried out correctly and the results are useful, e.g., always include positive and negative controls. Include the evaluation of appropriate controls during the experimental planning phase (see our RNA Lexicon chapter on Experimental and Data Analysis Planning for RNA-Seq).

• Ensure that functional checks on instruments and equipment maintenance are conducted periodically, e.g., check the functionality / temperature profile of PCR cyclers and ensure your pipets are calibrated.

• Perform laboratory environmental monitoring to meet equipment specifications (e.g., humidity, temperature). This is especially important for sensitive instruments such as sequencers.

• Monitor refrigerators and freezers containing any samples or reagents to prevent samples from spoiling due to power loss or equipment failure.

• Do not use expired reagents or reagents stored at the wrong temperature, as the functionality of the assay and the validity of results can be compromised.

• Make sure that there is enough space so that quality of work, cleanliness of the workplace, and safety of personnel are not compromised. Check these other important points for your lab environment and work space: