Extreme sensitivity of fitness to environmental conditions; lessons from #1BigBatch

Abstract

The phrase 'survival of the fittest' has become an iconic descriptor of how natural selection works. And yet, precisely measuring fitness, even for single-celled microbial populations growing in controlled laboratory conditions, remains a challenge. While numerous methods exist to perform these measurements, including recently developed methods utilizing DNA barcoding, all methods seem limited in their precision to differentiate strains with small fitness differences. This limit on precision is relevant in many fields, including the field of experimental evolution. In this study, we hone in on the factors that contribute to noisy fitness measurements and suggest solutions to avoid certain sources of noise. Surprisingly, even when common sources of technical noise are controlled for, we find that fitness measurements are still very noisy. Our data suggest that subtle environmental differences among replicates create substantial variation across fitness measurements. We conclude by providing best practices for obtaining precise fitness measurements and by discussing how these measurements should be interpreted given their extreme context dependence. This work was inspired by the scientific community who followed us and gave us tips as we live-tweeted a high-replicate fitness measurement experiment at #1BigBatch.

Best practices in designing, sequencing and identifying random DNA barcodes

“…In the aforementioned design, most such events (those that occur in the bulk of the fragment, between the inline indices) generate "inadmissible" index combinations that can be easily identified and discarded. Using this approach, we found that ~5% of reads had inadmissible index combinations (Venkataram et al, 2021), but others have reported rates of up to 43% (Kinsler et al, 2022). Note that, while it is possible to include inline indices in the one-step PCR setup, their utility would be limited.…”

“…In this setup, sample information can be encoded by a combination of four indices (two Illumina and two inline). In principle, samples tagged with different inline indices during the first PCR can be pooled together for the second PCR, although we do not recommend this practice due to the possibility of template switching events (Kinsler et al, 2022) .…”

“…One redundant design that we found particularly useful is where each 5' inline index is associated with a unique 3' Illumina index and each 3' inline index is associated with a unique 5' Illumina index. Such redundancy can be used to effectively detect primer cross-contamination, "index hopping", and template switching events that can occur during library preparation or on the Illumina flow cell (Illumina, 2017;Guenay-Greunke et al, 2021;Kinsler et al, 2022). These processes generate chimeric sequences, which introduce demultiplexing errors that in turn translate into errors in lineage frequency estimates.…”

“…They cannot expand the multiplexing capacity, but can help detect some index hopping events (those that occur between the Illumina index and the inline index that are on the same primer). The rate of index hopping is much higher on "patterned flow cell" Illumina machines, so we also recommend using a non-patterned flow cell machine for barcode sequencing whenever possible (Illumina, 2017;Guenay-Greunke et al, 2021;Kinsler et al, 2022).…”

Compensatory adaptation does not fully alleviate the costs associated with rifampicin resistance and occurs predominantly through off-target mutations

“…Additionally, we were unable to see a significant increase in fitness using either test for a clone carrying the YhjY S21S putative compensatory mutation, despite this mutation occurring in an extremely convergent manner across five independently evolving populations. Such apparent discrepancies may stem from the noisiness of the measurements themselves but likely often stem from the complexity of relative fitness itself (24). Relative fitness is likely strongly dependent on context and is the result of a complex interplay of factors.…”