NIH researchers develop lab method to enhance scientific reproducibility efforts

Scientists at the National Institutes of Health (NIH) have developed a method of tracking genetic variants in basic lab studies that allows researchers to do genetic experiments on complicated genotypes, or the sets of genes in an organism’s DNA which are responsible for a particular trait, using hundreds of replicates. In addition to providing a high degree of replication, the new method minimizes the chances of artefactual results arising from confounding background mutations or environmental factors.

“We’ve addressed two significant issues faced by genetic researchers with this model,” said Meru Sadhu, Ph.D., study author and scientist at NIH. “We’ve made it easy to track a lot of replicate samples in experiments studying genetic variants in more than one gene, which will help researchers see if their observations are repeatable. Also, we’ve generated replicates in a way that avoids them sharing background mutations that are absent from the other genotypes in the sample; shared background mutations are a problem because they can cause traits that the researcher will mistakenly think are due to the genetic variants they’re studying.”

Many biological experiments involve studying trait differences caused by genetic modifications, including genotypes composed of modifications at more than one gene. However, as the number and complexity of the genotypes increases, independently generating and tracking the necessary number of biological replicate samples becomes a major challenge.

A major development in genetic studies of large numbers of genotypes has been the use of barcode tracking in a pooled format. Inspired by this technology, NIH researchers developed a barcode-based method to track a small number of genotypes with large numbers of independent genetic replicates, enabling robust detection of subtle trait, or phenotypic, differences. The scientists used circular DNA molecules known as plasmids, which are found in bacteria and are used in recombinant DNA research to transfer genes between cells. They combine gene variants of interest that have associated DNA barcodes together to generate a pool of plasmids that each contain variants of multiple genes of interest, and a combined barcode that specifies the genotype of all the genes. The combined barcode also encodes a random sequence for tracking individual replicates, so the number of replicates can be easily increased by generating a larger plasmid pool. Sequencing of the pool of barcodes by next-generation sequencing allows the whole population to be studied in a single flask, enabling a high degree of replication even for complex genotypes.

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