Differential Selection
Activity
Enrichment
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Illumina Sequencing , Deep Mutational Scanning , Viral Competition
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The ‘‘differential selection’’ that the antibody exerts on a mutation is defined as the logarithm of that mutation’s enrichment in the antibody-selected condition relative to the control.
To determine the frequency of each mutation in the antibody-selected and non-selected conditions, we utilized a barcoded subamplicon Illumina deep sequencing approach as previously described (Doud and Bloom, 2016; Haddox et al., 2016). This approach uses unique molecular identifiers to distinguish true mutation from sequencing errors. It reduced the error rate when sequencing wild-type proviral plasmid to 1.5E-4 mutations per codon. KOD Hot Start Master Mix (EMD Millipore, 71842) was used for each PCR reaction. PCR products were cleaned with Agencourt AMPure XP beads (Beckman Coulter, A63880) using a bead-to-sample ratio of 1.0 and quantified via Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies, P7589) between each step in the library preparation process. Briefly, env was amplified from 20 mL of nonintegrated viral cDNA in a 50 mL reaction using the following thermocycler conditions:
1. 95C, 2 min
2. 95C, 20 s
3. 70C, 1 s
4. 60C, 10 s
5. 70C, 2 min 4 s
6. Go to 2, repeat 27 times
7. Hold at 4C
Next, 4 ng of env template was added to a 20 mL reaction supplemented with an additional 1 mM MgCl2. This reaction was performed independently for each of the 6 subamplicons for each sample. This PCR reaction was:
1. 95C, 2 min
2. 95C, 20 s
3. 70C, 1 s
4. 59C, 10 s
Cell Host & Microbe 21, 777–787.e1–e4, June 14, 2017 e2
5. 70C, 10 s
6. Go to 2, repeat 10 times
7. 95C for 1 min
8. Hold at 4C
Next, ssDNA molecules were bottlenecked such that each molecule would be read, on average, 2.7 times during Illumina sequencing. The 6 subamplicons for a single sample were then pooled, and a 20 mL PCR reaction, supplemented with an additional 1 mM MgCl2, was performed to add the remainder of the Illumina sequencing adapters. This PCR reaction was:
1. 95C, 2 min
2. 95C, 20 s
3. 70C, 1 s
4. 60C, 10 s
5. 70C, 10 s
6. Go to 2, repeat 23 times
7. Hold at 4C
Finally, samples were pooled, purified by gel electrophoresis, and sequenced on an Illumina HiSeq or MiSeq using 2x250 bp paired-end reads.