Amino acid preference

Growth/Fitness

Frequency of Occurrence

unitless

Illumina Sequencing , Deep Mutational Scanning

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The preference π(r,a) of site r for amino-acid a represents the expected frequency of that amino acid in a hypothetical library where each amino-acid is introduced at equal frequency.

In this experiment, each position of the WSN-HA coding sequence is mutated to specify all possible amino acids creating a pool of mutated HA sequences. To identify HA mutations that are tolerated during viral growth, this plasmid pool is used in tissue-culture to grow A/WSN/33 viruses with mutated HA using the reverse genetics system. The HA mutant virus pool is then passaged through cells once at a low multiplicity of infection (MOI of 0.1). The low MOI ensures that not more than one virus infects a given cell in the initial round of infection. This avoids a mismatch between the packaged vRNA and HA protein species expressed on the surface of the viruses produced from the cell (genotype-phenotype mismatch).

Comparing HA sequences from mutant virus pools (mutvirus sample) to HA sequences in the mutated plasmid pool (mutDNA sample) by 50-bp, paired end, Illumina sequencing yields information on the amino acids preferred at each position of WSN-HA when subjected to viral growth selection.

In addition, as a control to decipher baseline error rates during viral growth, sample processing and deep-sequencing, un-mutated WSN-HA plasmid (DNA sample) is used to grow wild-type A/WSN/33 virus (virus sample) and included in the deep-sequencing analysis.

Thus, the samples used for Illumina deep-sequencing are:

DNA : Made by low-cycle number PCR amplification of wild-type WSN-HA. Measure of error-rate in deep-sequencing.
mutDNA : Made by low-cycle number PCR amplification of HA from mutant HA plasmid pool. Measure of mutations in the mutant HA plasmid pool.
virus : Made by reverse-transcription of vRNA from wild-type A/WSN/33 virus, followed by PCR amplification of cDNA. Measure of error-rate during viral growth and reverse-transcription.
mutvirus : Made by reverse-transcription of vRNA from mutant virus pool, followed by PCR amplification of cDNA.Measure of mutations that are tolerated during viral growth selection.
Three biological replicates of each sample are made starting from independent wild-type and mutant HA plasmid pools. A fourth technical repeat consists of samples processed independently for deep-sequencing (starting from tagmentation). The samples for each replicate are multiplexed with Nextera primers for deep-sequencing and each replicate was run on a separate Illumina sequencing lane. Thus, the replicates used for Illumina deep-sequencing are:

#1 (or replicate 1) : DNA, mutDNA, virus and mutvirus samples made from one WSN-HA plasmid preparation.
#2 (or replicate 2) : DNA, mutDNA, virus and mutvirus samples made from another WSN-HA plasmid preparation.
#3 (or replicate 3) : DNA, mutDNA, virus and mutvirus samples made from a third WSN-HA plasmid preparation.
#1 repeat (or replicate 1 repeat) : DNA, mutDNA, virus and mutvirus samples from replicate 1 independently processed for deep-sequencing.