www.pnas.org/cgi/doi/10.1073/pnas. 115

freq

uenc

y pe

r 106 C

D4+ T

cel

ls

10-3

10-2

10-1

100

101

Q2 VOA

Intact

P<0.001

wk-2 wk12

92429243924492469247924192529255

Fig. S1. Comparisons of frequencies of intact proviruses and inducible proviruses. Scatter plot showing the frequencies of intact proviruses and inducible proviruses measured by NFL sequencing and Q2VOA, respectively. Each dot represents a different participant. Horizontal bars indicate median values. Statistical significance was determined using two-tailed Mann-Whitney U test.

1813512

1.11%

0.22%

0.03%

3.01%

1.37%

0.21%

0.02%

5.36%

1.35%

0.15%

0.04%

6.36%

1.45%

0.21%

0.008%

4.51%

2.12%

0.87%

0.004%

3.05%2.03%

0.55%

0.06%

4.09%3.24%

0.21%

0.10%

9.15%

1.56%

0.26%

0.13%

4.73%

0.74%

0.08%

0.05%

2.77%

1.37%

0.11%

0.007%

3.92%

2.21%

0.36%

0.03%

5.63%

9242 9243 9244 9246 9247 9241 9252 9255 9245 9249 9253

0.001

0.01

0.1

1

10

100

norm

aliz

ed p

erce

ntag

e of

pro

viru

ses

categorygagenvfull_sizeintactinducible

Fig. S2. Comparisons of different reservoir measurements. Bar graph showing the proportion of env+ proviruses, near full size proviruses, intact proviruses, and inducible proviruses within gag+ proviruses population.

0

2

4

6

8

Q2 VOA

NFL

Ave

rage

pai

rwis

e di

stan

ce

wk-2 wk12

92429243924492469247924192529255

9245

92539249

9254

P=0.79

Fig. S3. HIV-1 env sequences diversity.Scatter plot showing HIV-1 env sequence diversity from Q2VOA or NFL sequencing measured by average pairwise nucleotide distance. Each dot represents a different participant. Horizontal bars indicate median values. Statistical significance was determined using two-tailed Mann-Whitney U test.

0.004 0.004 0.004 0.004

0.001 0.004 0.5 0.004

92469242 9244 9247 9241

92459254 9255 9249 9253

0.004

0.02

Fig. S4. Phylogenetic trees of env sequences. Maximum-likelihood phylogenetic trees of env sequences obtained from Q2VOA at the pre-infusion (green) and week 12 (blue), NFL at the pre-infusion (purple) and week 12 (orange), and rebound viruses from SGA or outgrowth cultures (red).

0 50 100 1500

50

100

150

% env clonality

% o

verla

p w

ith Q

2 VO

A 924292439244924692479241925292559245

92539249

r = 0.808P = 0.0015

Fig. S5. Correlation between shared sequences from two methods and clonality. Scatter plot showing Pearson correlation between the percentage of clones and percentage of overlap between Q2VOA and NFL sequencing. Each dot represents a different participant.

NFL

Q2 VOA

106

107

108

109

num

ber o

f CD

4+ T c

ells

test

ed

wk-2 wk12

924292439244924692479241925292559245

92539249

P<0.0001

Fig. S6. Number of CD4+ T cells tested. Scatter plot showing the number of CD4+ T cells tested in Q2VOA and NFL sequencing. Each dot represents a different participant. Horizontal bars indicate median values. Statistical significance was determined using two-tailed Mann-Whitney U test.

9242

9243

9244

9245

9246

9247

9241

9249

9252

9253

Freq

uenc

y

Number of mutations

Predicted mutation accumulationHamming distanceDistance including possibility of recombination

Fig. S7. Relationship between latent and rebound sequences.Histograms show the proportion of env sequences (y-axis) and number of mutations (x-axis). The blue bars represent the Hamming distance between rebound and NFL/Q2VOA sequences. The grey bars represent the predicted distance between rebound and NFL/Q2VOA sequences based on a simulation of mutation accumulation during the ATI period for each participant. Yellow bars represent the predicted distance between the rebound and NFL/ Q2VOA sequences when the possibility of recombination is included.

ORFs intact?

no

packaging signal/MSD intact?

intact packaging signal/MSD deletions/mutations

yes no

hypermutated?

hypermutation indel/nonsense

yes no

yes

Fig. S8. Process used to identify intact and defective proviruses. Assembled sequences were aligned to the HXB2 genome to identify premature stop codons, out-of-frame insertions or deletions (indel), or packaging signal (Ψ) and the major splice donor (MSD) site deletions and mutations.