Supplemental Digital Content 1. Table: Primers Table 1. Primer-ID cDNA Primers Primer ID Barcode* Sequence† V3R_Buni GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNCAGTCCATTTTGCTCTACTAATGTT ACAATGTGC V3R_Buni_11 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNCAGTCCATTTTGCTCTACTAATG TTACAATGTGC V3R_001 ATAGCG GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNCGCTATCCATTTTGCTCTACTAATG V3R_002 TAGTCC GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNGGACTACCATTTTGCTCTACTAATG V3R_003 GCGATA GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNTATCGCCCATTTTGCTCTACTAATG V3R_004 AGTAGC GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNGCTACTCCATTTTGCTCTACTAATG *Barcoded cDNA primers were used for viral samples of chronic patients C1, C2, C7, C8, and C12. †The first random nucleotide within the Primer ID region containing either 9 or 11 random nucleotide sequences was used to buffer sequencing quality.

Supplemental Digital Content 2. Methods: Arbitrary Abundance Cut-off Primer IDs with multiple sequence reads detected in each well were used to create a consensus sequence for each original RNA template. The number of consensus sequences representing individual viral lineages detected in each well was converted into percentages. Then, the percentage of each viral lineage was assigned into one of 40 percentage bins that cover the percentages ranging from 0.01% to 100% with an equal interval between bins. For example, a single viral lineage consisting of 1000 identical consensus sequences out of a total of 2000 consensus sequences detected in a well will be assigned to a bin corresponding to 50%. Likewise, a viral lineage with 10 consensus sequences will be assigned to a bin corresponding to 0.5%. All viral lineages showing less than 0.01% was assigned to a bin of 0.01%. The number of viral lineages in each bin was counted and used to plot against the corresponding percentage of the bin to determine arbitrary abundance cut-off for the minimum abundance of individual consensus sequences.

  Supplemental Digital Content 3. Figure: Schematic Diagram of VOA-UDSA Induction of proviruses in resting CD4+ T cells   Deep Sequencing analysis to determine DVL VL1 VL4 VL2 VL3 VL5 DVL1 DVL2 DVL3 Individual DVL titer analysis IUPM The viral samples derived from cultured resting CD4+ T cells scored as HIV-1 positive were assigned with a specific Barcode and subjected to sequence analysis of the V1-V3 region of HIV-1 env sequences. All viral lineages (VL) detected in the wells analyzed by VOA-UDSA were used to generate a neighbor joining phylogenetic tree to determine distinct viral lineages (DVLs). DVLs are also determined by using in-house pipeline available at https://github.com/SwanstromLab/DVL. The number of wells expressing an identical DVL was counted for individual DVL titer analysis. The IUPM reported as the sum of all DVL titers per million resting CD4+ T cells was estimated using the maximum likelihood assuming the number of infected cells per well followed a Poisson distribution for each DVL.

Figure 4 1859 1 7 1697 Abundance 200 400 600 Base Number 200 400 600 Base Number Parental Sequence-A Parental Sequence-B Recombinant Point Mutation A: Green, T: Red, G: Orange, C: Light Blue, IUPAC: Dark Blue, Gaps: Gray Figure 4