Nature Methods: doi:10.1038/nmeth...barcode. Bottlenecks were simulat ed by multinomial sampling with replacement and we used equations (1) and (2) from Krimbas & Tsakas 5 to determine

Nature Methods: doi:10.1038/nmeth.3253

Supplementary Figure 1

Simulation demonstrating that calculation of Ne using a large number of sequence tags provides an accurate high-resolution estimation of Nb.

(a-b) We simulate a population with 5, 50, 500, and 108 tags present in equal frequencies. Then they were passed through a bottleneck

that reduced the population to 101-10

6 (bottleneck population size). In case of the 5, 50, and 500 tag simulations, this was followed by a

second sampling step (5x105) that fits the number of sequenced barcodes. 10

8 tags represent the ideal case where (virtually) each

bacterium has a unique tag such that after passage through the simulated bottleneck each bacterium is expected to have a distinct barcode. Bottlenecks were simulated by multinomial sampling with replacement and we used equations (1) and (2) from Krimbas & Tsakas

5 to determine Nb. The results of 1000 independent simulations are shown. To illustrate the relative deviation from the

theoretically expected Nb, data are normalized to the simulated bottleneck and the red, dotted line indicates the theoretically expected Nb (shown at 100 %). (a) In this box plot the median (black line), interquartile range (box), and 95 % confidence interval (whiskers) are indicated. (b) The scale is changed so that outliers (black squares) can be visualized. Note that the median of 1000 independent simulations accurately predicts Nb even with only 5 tags; however, the wide distribution of data-points make Nb estimations from this few tags inaccurate or impossible (negative values) with small numbers of experiments (i.e., few animal infections).



Schematic overview of the experimental setup and the Nb analysis pipeline.



The sequence tags are selectively neutral and stably integrated into the genome during the course of the experiment.

(a) Growth curves reveal the neutrality of the tags. V. cholerae strains containing different tags (pSoA158.1-pSoA158.32; black lines) were grown in LB medium with selection for the barcode (LB-Carb-Strep) and the absorbance at 600 nm was recorded in 10 min intervals for 20 h. The wild type (C6706; red line) grown in LB-Strep is given as a reference. (b) Same as in a without selection for the barcode (LB-Strep). (c) The stability of tag insertion was tested by comparing the cfu of V. cholerae grown in liquid culture without selection for the tags (LB-Strep) for 20 h on agar plates without selection for the tag (LB-Carb-Strep) and plates with selection for the tag (LB-Strep). To control for the technical variability of the assay, the same culture was also grown twice on plates without selection for the tag (LB-Strep). No significant difference (p = 0.30; Wilcoxon rank sum test) was detected between both assays. Individual tags (pSoA158.1-pSoA158.7) were tested in biologically independent triplicates. The bold line indicates the overall median for the indicated condition; the dotted line highlights the expected 1:1 ratio.



Determination of the optimal theoretical framework and similarity threshold for calculation of bottleneck population sizes.

Correlation between experimentally determined bottleneck population size (bacterial load) and estimated bottleneck population size (Nb) with methods from Krimbas & Tsakas

5 (black symbols), Nei & Tajima

6 (middle grey symbols), and Pollak

7 (light grey symbols). The

diamond, square and triangle symbols represent biologically independent replicas. Each graph uses the same sequencing data that have been clustered with different similarity thresholds (Sim. threshold) during tag enumeration with uclust. The thresholds are given in the header of each graph. A sequence similarity threshold of 1.0 produced negative Nb for some data-points which are not displayed in the graph. Note that methods from Nei & Tajima and Pollak produce very similar results so that the symbols overlap. The same dataset analyzed according to Krimbas & Tsakas, with sequence similarity threshold of 0.9 and after INOC54 correction is given in figure 1a and used as a calibration curve for the animal experiments. The INOC54 correction removed non-specific tags, but had minimal influence on the results which indicates that the Nb determination is very robust and can tolerate the loss of several tags.



High–spatial resolution analysis of bottleneck populations sizes in the proximal SI.

An additional representative example (Fig. 1b) of bottleneck population size (Nb’, black dots) and bacterial load (cfu, red squares) at 20h post-infection throughout the gastro-intestinal tract of a single animal after infection with 10

9 tagged V. cholerae. The dashed, grey

lines mark the resolution limit for Nb’ estimation. The sampling sites are indicated in light red in the schematic diagram of the gastro-intestinal tract



The recolonization of the proximal SI is not caused by coprophagia.

To exclude the possibility that the increase in the founding population in the proximal small intestine, which occurs in the late phase of infection, is caused by the uptake of tagged V. cholerae in food or stool, rabbits were prevented from food intake after infection by fitting them with a pet cone and housing them individually. Bottleneck population size (Nb’, black dots) and bacterial load (cfu, red squares) in the proximal (I1), middle (I2), distal small intestine (I3), cecal fluid (Cf) and colon (Co) of three infant rabbits from a single litter at 20 h post-infection infected with an infective dose of 10

9 cfu. The marked similarity of the Nb’ values shown here with those in figure 1b and

2a (late phase; pI1 = 0.71, pI2 = 0.67, pI3 = 0.83, pCf = 0.67, pCo = 0.49; Wilcoxon rank sum tests) argues against the idea that coprophagia is a primary explanation for the high Nb’ values in I1 during the late phase of infection. The dotted lines indicate the resolution limit for Nb’ estimation. The sample medians are represented by horizontal lines. Corresponding Nb’ and bacterial load from the same animal are aligned vertically and always in the same sequential arrangement throughout the sample loci. The sampling sites are indicated in red in the schematic diagram of the gastro-intestinal tract.



Onset of fluid accumulation in the GI tract during the late phase of the disease correlates with the backward movement of V. cholerae from the distal (I3) to the proximal (I1) SI.

The volume of fluid (black dots) accumulated in the cecum of 19 infant rabbits from 12 different litters (a proxy for the action of cholera toxin) was measured in the early, middle and late phases (~2 h; ~7 h; ~20 h post-infection) of infection. Sample medians arerepresented by horizontal lines.



High technical reproducibility of the sequencing analysis.

(a-d) Scatter plots of tag frequencies from different inocula and sequencing runs. All inocula cultures were started from aliquots of the same frozen library. Samples of the same inoculum culture (A and A’) were processed in parallel and sequenced on the same sequencer run (a). Samples from two independent inocula cultures (B and C) were processed independently and sequenced on the same sequencer run (b). Samples of the same inoculum culture (B and B’) were processed in parallel and sequenced on separate sequencer runs (c).Samples from two independent inocula cultures (A and B) were processed independently and sequenced on separate sequencer runs (d). The correlation coefficients of the linear regression (R

2) are given in the figure.

Primer # SequenceP9 GCAGGCAGTCTCGGTCAATA

P10 TTGTCTCATGAGCGGATACA

P47 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGTAAAACGACGGCCAGT

P48 CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P49 ACGCTCTTCCGATCTTGTAAAACGACGGCCAGT

P51 CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P52 CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P53 CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P54 CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P55 CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P56 CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P57 CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P58 CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P59 CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P60 CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P61 CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P62 CAAGCAGAAGACGGCATACGAGATTGTTGACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P63 CAAGCAGAAGACGGCATACGAGATACGGAACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P64 CAAGCAGAAGACGGCATACGAGATTCTGACATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P65 CAAGCAGAAGACGGCATACGAGATCGGGACGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P66 CAAGCAGAAGACGGCATACGAGATGTGCGGACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P67 CAAGCAGAAGACGGCATACGAGATCGTTTCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P68 CAAGCAGAAGACGGCATACGAGATAAGGCCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P69 CAAGCAGAAGACGGCATACGAGATTCCGAAACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P70 CAAGCAGAAGACGGCATACGAGATTACGTACGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P71 CAAGCAGAAGACGGCATACGAGATATCCACTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P72 CAAGCAGAAGACGGCATACGAGATATATCAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P73 CAAGCAGAAGACGGCATACGAGATAAAGGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTCTCATGAGCGGATACA

P74 AATGATACGGCGACCACCGAGAT

P75 CAAGCAGAAGACGGCATACGA

P78 CTGCAGATCTGCAGGTCGACGGATCCCAAGCTTCTTCTAGACCGGCTTACTAAAAGCCAGAT

P79 GGAGTCAAAACAAACTAGCGATCGAATTCCCGGGAGAGCTCTTATATTCCCCAGAACATCAG

P80 GGAGTCAAAACAAACTAGCGATCGAATTCCCGGGAGAGCTCCGCAAGTGGTTGTGGTAAAA

P110 TGCCACCTGCAGATCTGCAGGTCGACGGATCCCAAGCTTCTTCTAGACAGGAAACAGCTATGACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACTGGCCGTCGTTTTACACCTCAGCGGAGAAGAACACT


c.sanders

Typewritten Text

c.sanders

Typewritten Text

c.sanders

Typewritten Text

Supplementary Table 1

c.sanders

Typewritten Text

c.sanders

Typewritten Text

Tag number Tag sequence1 CTCGAGATAACATATGTATCACTAGTG

2 CGAAAACGAATCAGGAAAAATCAAAAAG

3 TTAAACATGACCACTAAACTAGTTACGCACG

4 TTAACACAAGGTAAGCCTAATATAGAAACTG

5 TGGGGAGGAAGACTTGCGTAGAATGTTTCCG

6 GACCTCTAACTATAAATTGCGGTGAGTTCTG

7 CCAAGGCTAAAATAAGAACTAACG

8 AAAAATCCCCGTGCAGATTGAAAAAAATCCG

9 CCAAGCGCCCATATTCGAGACATCACCGAGG

10 GAGGTTAGCCACCGCGAACTGAGATATAAGG

11 TAGAGACCTTAACCGGAAATGACGATGGGAG

12 ATTATGTGTATAAATCGACCTCCCGG

13 GGGATACAGCGCGACTGCCGGAGAGCCCTAG

14 GCTCCGGGGCAAGGACTAGGAGACCATCGGG

15 AATCGACTCGGCACCGGTCCATTAGCTGGTG

16 AATCGAAATGGCCATACCGACGGGGTTAAGG

17 AGACAGGAGTTTATCGCGGTAGAGATAGGGG

18 GCATCCGCAATCGCGTCGATTCGCTAAGTCG

19 ATCGCATGACGTGTCTCGACTGTGAAAATGG

20 GAGTTAATAAGGGGGCAGCGCGACGGACGCG

21 GGATTCTCCTTGACCATGAGTCATGAAGAG

22 GACGCGAAGGTAGTGACAGTCACATAAGAAG

23 ATGAATCGAGAAGCCCATGGCTGTTAAAGTG

24 CCGAGGGGCACAAAATGCACCAAATGTTTCG

25 AGTGGAATTACATGGTCGTGGTACGCCGTAG

26 CGGAGCACCGGAGTCGCCTTTACGTGAAGTG

27 GAGTAATCACCAAGTCAAACCTTAGACGCTG

28 CAGACAGACGGCGGGGCAAGAAAGCGATAGG

29 GCATAGTGGCGTACGGGCTTGCCTAACAATG

30 TAAGATGTAAGCGCGCAACGCCAATCAAGGG

31 ACGTAACTATCTGAGGCGTTAAAGTAGTCAG

32 CAATTAAGGCTCTTGAAGAGCAGAGTACCTG

33 CTTAGCACATATTGGCAGATTCGAGATACGG

34 AGGGTACCGCGAGTATGATTGCGAGCAGCAG

35 GCACCGCAAACGCCAGGCGATTCCCAAGGTG

36 TTAGGGCATATGCGGAGAACCATATTAAGTG

37 GCAGTACGAGTAGGAATGACAAACTTTAGAG

38 TTCAAGACCTCCGGGGAGAAAGATGCCCCGG

39 GTCTGGCTGTATGCCCTAAGATCG

40 GTTGTTTCAGCTAAGAGCAAATATCCTCACG

41 AAACCTCGCTGTCGCCTTGAATCCCACGTCG

42 GGCAGGGTCGATACAGTGCATCACTGGAAAG

43 AGAGCAGCGTATTGGACGATCCTGCCACAAG

44 TAGATCACCTAGAGCTCGCGTGCCTTGGAAG

45 AGAAAACCGAGGAACTTCGCGCTTCGATTGG

46 ATGGAAAGCATGGTAAAAGGTCCAGATAAG

47 TCTGACAAAAGTAAAAGG

48 ATGCACCAACGCCTTTGATTCATGCCGGTGG

49 AGAAAAGCAAGAGGTTGGACCGATCGTAAGG

50 GTAAAAGTAATTATAATAGACAGTCGAATTG

51 GTGGGTTGCCCCAAGGGCAGAACGAAGGATG

52 CCTAGATTACCTGATCAGAGTATGATCTCCG

53 AACATTACGCGGGTGTGTGTAATTCAAAACG

54 TCAAAACAGCACCAGGGCCGATAGGGAGGGG

55 GAGCTTGTTTAGAGGCTATTATCTTAGATAG

56 GCAATAAAGTAGTAGAAGGGCTGCCGTGGAG

57 GACCACGGACTACGAGGGGGCACTTCTAAGG

58 ACGACATGGAAGTAGGCTGCATACGAAGTG


c.sanders

Typewritten Text

Supplementary Table 2

c.sanders

Typewritten Text

59 AGATTATAAGCGGGGCGAAGAGTGTGTTATG

60 AATTACATCCCGGGCGATAGGTTTGAAGATG

61 CATCGCAATGGCAAAGTCAATATTTCCAGTG

62 TTGGGTCGACGTAATGCGCAACCTGCCATAG

63 CGACCAACACAAATCAGGGTGTGTACCACAG

64 ACTATTTCAGATACCTATTGTGACGCTGCAG

65 CACGACGAATGCGGCAATATACATAGCAGGG

66 TCCGCACACATCTACCTATATAGAAGAGGCG

67 CTGAGGTGATAGCGCACTGAAATATTGGCTG

68 CCGGGCACAATCAAATGGGTCAGAATGCTGG

69 CAATGTGCTAGAATCGGCACCAACGCTACAG

70 CCACACCCGTAGCGTACGGAAAGCCATCACG

71 GGAATTTACTCATTTGAAAGAGCTCGTTAGG

72 AGATACGGAGGTCCTCACAAGGACGTGGACG

73 GGAGAGACTGACAAGCAAGTCCGTAAACCCG

74 AATTCAGCATACGAAAAGATTGAAAGCATCG

75 AGGCTACTGAGAACACGTAGTTTGACTGACG

76 CAAAGGATTGAAGCTTCAGCACCTATCGCGG

77 CTGAAATACGGAAAGGAAATAGGCGATAAAG

78 ATAAGATTGTAAAATAAGGACAAGAGCATGG

79 TAGCAGGGAAATGACAGTGTGGTAG

80 CAGATTGAATAAAAATGCGGCCCAAATCATG

81 GCGATAGGGTCGACCCGATCCACGAGGGATG

82 ATGCGTATTACAAATCATGG

83 TGTAAAATACTAAGAGGACTGCTCGCGTCTG

84 AATAGTGTGGGAGTCGTTAGGGGCAAGAAGG

85 AACTTGGAGGTGTCAATCATTGTCCTCGGTG

86 ATCACTCATCGCAACCAAGG

87 CTGTTGCAGATGGAAGCCTACCCACGAAGAG

88 CAGGGCTATAAAAAAGCAATGCGGAGACACG

89 CCAGGTAACCACCGCTATGAACCTAGTACAG

90 GAATTGATGGTCCGGATTCGACGCACTTCTG

91 AGATTTTCGACGGAGTAGAAGTGGCAAATCG

92 AGAAGGATCAGCAGAGTG

93 ACGCGTGGGCTGTTAACGTGATCCCGACCCG

94 AATCAAATAAAAAGCCGAGGCGGTCACTCTG

95 TCTTCAGCAGCGAATGAGTCATTGTGTACG

96 TACGCTCCTACACGAAAGAGAAAAGTCCGGG

97 CCCAGGGAGACCCACAACCCACTGGACAAAG

98 GCAAGTGCGACCAACATTCGACGCCCAGTTG

99 TTTATAGCCGCACTCGAAAGGGGACGTACGG

100 AGGTGCACTCGGGGGGGATGTTACGGGATTG

101 GGAACAGCAAAATATCTATGCGTGAGGGAAG

102 GCGAGTGTAATCCGCTATACGAAAATAGCGG

103 GAATTCCAAGAACTACAATTTCTAACATCAG

104 CAATAGAGCCGACAACGGCGTAGATGCGAAG

105 GGAGGAATACAGAACGTAGCAAAACCACGCG

106 GAGTTAATCTCCGGGCACCGAATTAACCGAG

107 ATCTCACGCTGATGATACACCGAAAACTTGG

108 AATTCGTGCCCCGCGCGGGATAAATGCTTAG

109 AGTGCGTGAGGACGACGGAACCCGGAGAAAG

110 GGACTGATGGACACAAGATCGTAAAAGCATG

111 TGCTTTTCGGACATGAGAGAACCGATTACTG

112 CCCGGACCATTAGTACCAAACCCAACGCAGG

113 GGGGAAGTACCACCGTAAACGCGAACACATG

114 TCAACAAAAGCATATCACGCTTTTACGCATG

115 TCTCCTCGAAATGCCAACGAAAAAGGGCGGG

116 CCGGAGTGAACGTCTGCATACGACCTAGACG

117 ACAACAGTTTCAATACAAATAACCTAGCGGG


118 AGGGGAAGGAGCTGGGTGGGATATGCCGGCG

119 GGGAGCTATAAAACAATAGTTAGACACGCAG

120 TCCGTCAGCAATAGACGTGATTCCTTAGGTG

121 AGATGATTCAACCGCTCG

122 GAGGCTGAAAGCAGGGCAACCAATGGGCGCG

123 CACGAGAGGAAATAGCCCTTGCTACCGCGAG

124 CTGGGCATGATAATCGGTCAGAATTACAACG

125 GACCTTAATACCCACTAGAACGTACTTTGAG

126 GCAGGGAGTCAAACCATATGAACCGGTATCG

127 GTAGACGACAGGGGTTCGAGCAATAAACTAG

128 GCGAACAGGACGTGGGTTAAACCAGGAATGG

129 GTTTGGAAATATTGGGCGTTAGTGTTCATTG

130 CCAGAGGATCAAAAAGTGCGGACAATCTTAG

131 CCCTCCAACAGGGTAGAAGAAGAGCCACTAG

132 AACAGGAAGGAATGTACACAATCAGGTTATG

133 AACATAACGCGCAAAAGTACTTCTACGTGGG

134 GGGCAGATTCGGCGTTAGTAAAATCTCATGG

135 CCACATGTCCAAACAGATTAACGGGTGCGCG

136 GAAACGGGAGTTTACAGAATAATGCTCCTAG

137 TTAACAAAGCGACCTGCCAGGCGAGTGCCAG

138 GATTACTGATTCAAAACCGAGGAGACTCG

139 AAATTTTTTGATATCGAAGGAGTTCCAATGG

140 TCATTTTGTAACAAATTCTGCATGCAAAGCG

141 CTCCGAGCCCCTAGGGAACTGCATCTGCTAG

142 TCGAGAGGAGCCCTGACAGGAGGGGGATTAG

143 GAATTCACGACGAAGAAAAGACTGGAAATGG

144 TGCTGGTGTTACGTCTAAGTGTGAAACACGG

145 CCAATTTCGTGACTCTTAAAGGGAAGGCGGG

146 TGTGCGAGGAGGCAGATCTCCCAAAGCCTTG

147 TAAAATCGGAGCATGCTGGTCCACTAAAACG

148 GTTTCTGACGAGTGGTACGTAGTGGATTTCG

149 TGGCGCCAGTTAGGACACGGACACACGCGGG

150 CAGAAATATTATAACTCTTTAGAGCGTCATG

151 AAAAACTAGACGACAGCCGGATGCAGATTGG

152 GACGATCGCATAGGATACTCCCCCGAAAGGG

153 ATTACCCTTAGGTCCATGGAGTCCCGGGGGG

154 GAACACGACGGTATGGACGCGAGAGCAGACG

155 CACGGAAACTAGAAAGCGGGTAAGATAGAGG

156 AGATTAGCGGGACGAAGACTCCTGACAAACG

157 GGGTCGCATGATTGGCACCATATAGCAATGG

158 TATACAAAACACTCTG

159 AGTGGAGGATGACCGTAACACACGGTTAAG

160 CTCCTGGACGGGGCATAAGTGCGACCAGACG

161 TATGAAATACCAGACGACATTAGTTGGATCG

162 TTTGCCGGTATACCCTAAGCACCTTGGAACG

163 ACACAGCATGGCGCAGATCCAAGAGAGTTGG

164 TAACGCAAATGACAAACCTAATTCAACACGG

165 TTCGCATCGCGCGCCAGCAAACTATGACTTG

166 AAAACGCTCATGTTCGGCAGAGGACCGATTG

167 CAGGGATCGGAAATTGTAGGTGCCAAAAGTG

168 ATGCACGCTTTGATTCATTGATAACTTTAAG

169 ATACGCAACGAAGTACAGTTATCCTAGCCGG

170 AGAAGTGTAAATAAACGATCTACGTACGTGG

171 TGAAACCAAGAGACGGTCTAAGGACAACTAG

172 GAACGTTTAGTGAGAAAGGAAGGACGAAGG

173 CGATAACCTGTTTATCGCCGTAACAATGTTG

174 GTGAGCCCAGCAGTCGAAGCAATATAGGTAG

175 TAATCCATCATTCCAACTAGGCATGGGCTTG

176 ATACAAACGCGGGAAAGCGCAGGCATCTTTG


177 AGTCTAGCATTATCAGCAGGCGGACCGCCAG

178 AAGCACTACTGAAGCTAGTCTCATATGATGG

179 CTGGGAAATCCATAACGGCGCGCGTAACTAG

180 ATAAGGTTGTGAATAGTGAAATATTTTGCTG

181 CCCGGTATTGTCATCTGGAGTAGTCTACGTG

182 AAATCATCAAGCGTGAAAAAATTCGCACACG

183 AATAGTTGGATCACGCAGGAGTCAAACTAAG

184 AGTATTACAAGTGCGATCTTCAGTACTCCCG

185 TTTGTAATAGGGCACAAAGGGGTCG

186 GTTAAGCCAACGGCCGAGACCGTATTGGTGG

187 AGAGGGAAATTACCTAAAGTCGAGTTAAAAG

188 CACGGGGATCTGTCGCATCGCGTGATGTGGG

189 AACTGTGCATTTGGAAGACGAACACATGG

190 TAGCGCTTCAGTTTGATAGATGAGTTGTGCG

191 AGGATAGTCAGAAGCGTGAACTATTTAGGAG

192 ATTTACGCTGGGCATACGCATCCGCGCCCAG

193 GCGTACCGAAGCATCCAGAGTCGCATTGAAG

194 GAAGACCAGCAATCTGCGTACTACATAAACG

195 TTGCTACCCTAAGCCACAGTTGGGCCAGAAG

196 CACCCTCCCAGAAAATGGAGCAAATGCCCTG

197 GAAAATCGTGCGTACTCTGTAACGCAGTCAG

198 GGATTGTATAATGTAACATGGTGAGGAATCG

199 ACAGGTACCGATGGTGTATTTAGGCAGTGGG

200 GCTGTACTCGTGATCACTCGTGAAATCTCAG

201 CTTGGCTTTGGCACAACTGAATTCTATGTAG

202 ACATACGGTTACGAACTCAGCAGCTAGGCTG

203 CATCCTGAGTTGGGAGTAGCGCGGTCCAACG

204 ATAAAGGGATCGGGATAGCATGTGACGCGGG

205 CCAACAAGCATGCCCCATATCCAAGACCCCG

206 TCACGAACGGAAATAGAGCAGGTGCCGCGGG

207 ATGGTCCTAACTGCGAGACGGAAATCCCCAG

208 AGAGAGTGATAGGACATATCGGGAGTCAGCG

209 GGGACTTTGGTACTAAATTTAGAGCAAGTAG

210 TTCGATGGTCTACTATAAAAGAGGAAATAAG

211 ACCGATCATTATGAGAAGTGAAATAAATGAG

212 TCGGCAAAAAGCAGCAGAATCAAAACAACGG

213 CAATGAAGACTCTGGTTTTAAACGATGGATG

214 TTCTACGAGGTCCTGCGTTTAATACGTACGG

215 GGATATGGGCCTGGATTATCCTTACAGGGGG

216 CCGGGGGAGAGAGTTCAATGCAGCCTGATGG

217 AATTCAACATAAGGGGCACACTTGAAATCAG

218 CTGTCGTCTGGAAGGGGATCGAACTTTACCG

219 CACACCCTCTAACAAGGTTGACTAACTGAAG

220 GCTAAAGCGCAGTGTTTAACGCGAGGGTCGG

221 GTGGGTCCCCCTGTGTTTGAGGGTACCAAGG

222 AAGCTACCCACTCGAAAACGGCGAAGCGAG

223 ACGTGAGTTCCAGTAATCAGTAAAACTAGCG

224 GGGTTGCACAAAGTTAACGGTTTTTCTTTAG

225 AGACCAAAATGGGTCTCCCGAGTTATCCGTG

226 AATAGAATAGAACACAACGTGTACCTCTTTG

227 TACCCCCGTAGCGACAAGAGCCATAGGACCG

228 AGAGCAAGGTCACCACTGCGAAATGACCACG

229 CAAGAGTGCACCGACTATGCGGCAATTTCAG

230 CACGGAAATCGTTAAGTGTAATACGAGAAAG

231 ATAGTAGGAGAGGATAAATATGACTACACCG

232 CTAATAAAATAAGCTTAACACCCTGCGCAGG

233 GGAGGCACCAGCAAAACTCAAGCCAGGAGCG

234 GAGCATGGGAAACCTAAGGCAAGACCTCACG

235 CAGGAGAAGGAAGGAGAGTGCGGCTCCCCAG


236 ATGAAGAACTAGTCGGGGGGCTCGAGAGGAG

237 GAAACGAACATAGATCCTCGGCAAACTGGGG

238 TAGGATCGTAAGGGGTAACACTACCAGCGGG

239 TGAAACCCCGAGGACCACCACGCGAAGGAAG

240 TCGCCTACGAGGGTGCGGACTTTTACACTAG

241 AGTTTCCTAAAACATTACTGATTGACGGTGG

242 TGTACGGACGGGAAAGCCGCAGCCTGCAATG

243 CCTAGGGCGGATCATTCATAATTGCGTTAAG

244 AATATCACAGAGTCTAAGCTAATCTATGCCG

245 CGGACCGAATGTTGGTAGTACCAGGTCGG

246 GAAAAGCTAAAGTACCCGAGACCACTGCCAG

247 AGTAAGCAAGGGAATTTGGGTGGTGCCTCAG

248 AGCGGTGAAATAGAGCAGGTGTGGATGATAG

249 CTGCAGCGCTGGTGGGTGGGCAGGGTCAACG

250 GGGGAAAAATATTACTAGAACATAAGCCAG

251 TACCGAGAGCGGCGAATGTGAAGCATCAAAG

252 GGCGAGAATGACGCAGGGGTGCGTCTACTAG

253 TAGCCGGAGACTCAGCATGAGTAGCGAATAG

254 CAGGATATGATGCCCCACACCAGCGTAAATG

255 AAAAAGACATGAGGATCAGCAAGAGTGGCTG

256 GAACCGCATAAATGTTTTGCGCGCGATATAG

257 TACACAATGAGGCATAAGCAAGCGCAAGGCG

258 ATAGTAGCACGGCGCTTGGCAGCTTTAATAG

259 AAAACTCTCGGTAACATAAGCACAAGCGATG

260 CAACAAAGGGGATAAGCGGAGCCTTCTCAGG

261 AGGAAAGAAAGGCTAAAAATCGACACACCCG

262 ACCGTGAGGTATCTGTTATCAGCTAGATTAG

263 ACATGGAAAGAAGTAATGAGTCTCCGAGCGG

264 CTTGTGAGCTATTGTCGCCGGGGACGAGGCG

265 GAAGATGGCCCAAGATTGAGCGGCATGATCG

266 TGCTAAGGGACTGCCTGCCCCAAATGAGAAG

267 GAGGGCACTGTAAGATGGTGACAGATAGATG

268 GGGGGCCCGCGCTCAATTGGCGCGAAGGCAG

269 AATAAAATCTCGGGAAAAGGCTCAGACCGTG

270 TGAACACCGGGAAATACCGCCAATTCGATAG

271 CCACCCGAAAGTTTCTTTATATTGGACCAGG

272 TAATTATGGTGGCTGAGGACCAAACACGAAG

273 GACAAGTCCACGAAAGACCGTTGACGATCGG

274 GACCGAAGTAAAGATAGTTCGTAGAAACTGG

275 TTTTTTGCCCAATGCGCTGATTGTGTCCGAG

276 TCGCCACCTAAACCGACGCTTGAACCAGCGG

277 TATGTAATTGGTCAAGAGTTAAGAACTAAGG

278 TGCCTTGATATTACGGATACAGGGTACAAGG

279 AATGTTTGAGGTACAAGTGCCAGGCTAACTG

280 AATCAGTACCGCCAGAAATATCTCCTTGCGG

281 CAATCTCTGAAAATCAGCGGCCCATACTTGG

282 AGTAACAAATCGCAAGAGTTTTAAAGCTGCG

283 GGCAGCAAATGGGGTGGAAGACTTGCATGG

284 CACCTAGGGGGAAGTGCTAGCGACTGACAGG

285 TGAATCGAGGAATTCTTGCATAACATAACCG

286 CTAGCTCATTCGACGCTACTCCTAAACCTAG

287 ATGTAGCAATGTATCGGAGCAGTCCACACGG

288 GAGGGAGGGGTAAGCCTGACAAAACGG

289 AGACTTCGCAAGCAGGATCGCAATGCACACG

290 AAGTTGCAGGAGCAACGGAAACACATCCCGG

291 GAAAGCATACGCACGCCGGGACGCGTAGTTG

292 AAAGGAGATGTGTCCTGTCGGACGGCGAATG

293 TGAGATGAATCGGATGAAAGTAAGATGGAGG

294 GCTAGGGGTTTTTCTCCCCTTAAAGACCATG


295 TCCCGTAGCTGGACAACCACCCCTCCAACCG

296 ATATATACTGCTTATGGGTAGGAGCG

297 GTGAACCAGGAGTCCTAACTTAAACCGGCGG

298 AAGGGTTTTCTAGCACCTTGGGTCTGTGTGG

299 AATACCAACCACATATATAAAGGACAACTCG

300 GAGATGACCCCTGCCGGTAAAACCTTTGTGG

301 TGAGTATGGCTCGTATCAGCGCTACTTCTAG

302 CTCAGGAACCAAAGCGAGAAAAGCATATTAG

303 CCAGGGCCGGGTCGGGTAAGAAG

304 GGTTAGAAGATTAAGCGTATACGTCTGCGGG

305 ACCATGGTGGACGTAGAGCACGGCAGACTTG

306 ATTGCCGGAGAAAATAGGGGCACCATTTCCG

307 CATACGCCAGAACCAATCACTGCCAAAAACG

308 AATCGAGGAGGAACCACCAATTACCCAAACG

309 CCAAAAAAGCGCTGAGTTCTTCACCTCATTG

310 CGAAATTTAGTATCATCCCCGTCCCAGGTGG

311 ACTCATGGTATGAGTGATCTCGGATGCTCAG

312 AAATGTGCTGACAAGCGACTGCTGAAAGTGG

313 GATGCGCTGGATGCTTAAAACGCTATGGCGG

314 AGACCAAAGGTTATCCGAAGCTCGCCATGGG

315 TAGCGAAGTCAACGTACAAACATTCCGGGG

316 TGGAGAATAGTATCGTCAAAGGCCCTACAAG

317 ATACTAGCCTAGGAAACGTGGAACACCTGTG

318 GCCACAAAGGATGCTCGAGAGTACTCAGCTG

319 ACGCGGGTGATATGACCCAATTTCTAATTGG

320 CCTAAGAGGATATTGGGCGAAGATAGGGTCG

321 ATTATGAAGGATGACCGAGCCCGTCCAATTG

322 TCCAGGTTAACTTAGAGCCCAGAATCTGATG

323 ACGGTTGCTTGAGTATGGTGCATCCGGTAAG

324 ACATTAGCATCTCAGAGGGGTAAATGGATTG

325 AGGAGTTATCAGATAGGTAAAGGCTAACGCG

326 CTCGCGGGTCTGTCTGACGCTTGGGGCTTTG

327 GCTGTATTCGTGGCAAGCAACTTCGTTGGCG

328 TTGCTTCCGGCATGAAGTAAAGAAGATCTGG

329 GGATGTAGAAGTGCCGCAGTGATTGAACAAG

330 CGCCCGAGAAGAACAGCCACAATCGAGGTTG

331 CCAGAGCGCGTTAAGACTCGAGCGTATAGCG

332 CCCAACGTTTGTGTACGAAGCAACCCTATTG

333 CGGTAGCAAAATTAGGGTGAGAAACGCTCAG

334 GGAAGAGGAGTCAACAGGGCGGCAATATAAG

335 ATAGCCTTCAACCCGATTGAGGGAATGTGGG

336 ATGCTACTCCTAAGTATTGTGGGGACCCTAG

337 AGACTCAACGTCAGTGGTGAGGGTAAGGGCG

338 AGGGGGGCACAAGGTCAAGGGAATGAAAGGG

339 TCATCCGTATAGTATGAAATTAGAAAAGATG

340 AGAATGCCATGGGGCCAGATGTAGGTGGGAG

341 CAAGTGAATGGCACCTATGATTAAATGAAAG

342 GAATTGATAAATCAGGCCCAAAAGCGATACG

343 GCTACTTCTGTCACCCATCAGGACGGGCGGG

344 CGCTTTCGGGGCTACGCCCATCTTAGACATG

345 AGCTAGGAAAACCCCCAACCGTGGGGGATCG

346 TACGCCAAGAAACATAAGATGGCGGGGCCCG

347 GCTGCATGGATAGACCTTCGCTTACCCCGGG

348 GTGACCGAACGCATAAACCATTTGTATTGGG

349 AAGCTAAACACACTACGACCACACCGATGAG

350 GACCGGTGGAGCATTGGATGTTTAGTAGTGG

351 GAGGACAACGGTATCTGCTACTTAGAACGGG

352 ACTGGCAAGATGTGCATCAAAACACAGTATG

353 ATACGAGGAGAACGAACAACCCCCGAGTGGG


354 ACCCGTACCGGGGGGCCTACACAAGCAATCG

355 AATGGTAGGTTGGAACTCTTGAGGAAGAGAG

356 TAATATCTAAAGTGGCGTATCGTGATAACGG

357 AAGGGACGCAATCATTCAAACGCAGAAAGCG

358 GAACCGCACAACGGTGAAGCTTATTAAGTAG

359 TACGTAGGATAGGGACCCGGAAGTTTGATAG

360 AGCCAGCAGACCCTACAAACCGCTGGGTGG

361 GCCAAGCCCGGTGAACCACGCATTGCATTTG

362 CTTTCATGAAAAGTATATCCTGAGAAGTGTG

363 CAAGCAGCGACCTTTCCGAAGAGGACTGAG

364 GTTGAACACAAAGTAAGCTATAAGCCGGCGG

365 TGGCTGTTAAACCACCGAAGCGCTTGTCTGG

366 CCATAATTTAAGCCATAAGAATTGATCTAAG

367 AACATCACAGGTTCCGAGCATGAAGCTATCG

368 TGAACTCACACTGGGCCAAAAGAATCAATCG

369 AAACACACGAGAAGGGATCTACCAATGACTG

370 CGAAGGCTTATCACCTCCGAACCTGAATTAG

371 GTGGTGACGCCAGGTGATAAACTTCTTCTGG

372 ACTGAATAGTTCAAATATTTAGTAGGGATGG

373 CAGTTTGATGCGGCTTTCAGATCACTAGATG

374 GCCGGGATGTTTGAATGTTGCGCCTTGCACG

375 TACTGCCAATACGGTTCACACCTGCAGAACG

376 GATCAAAAGCGGTGAAATCTAAGAGCGTATG

377 AGAAAACGAAATTTCTTTGTAGACCTGGAGG

378 CGAATAACTGAAAATTCCGAGG

379 GCCGCGGGAAGACAAGCGCCGGGGACAGCAG

380 TATATAATATTAAGAATAAAGTCCGCGCATG

381 AATTACACGGGGGTTTCGGTGACCTGTCGGG

382 ACATGAAGTTACTTAAACTGGATCAGGAGGG

383 CCTACAGCGCGAGGAGCTGACTAGTCAAGG

384 GAGGTGCAGGTGATG

385 GAAGATTTTTTTAAACGCACTGCAACGGCGG

386 ATCATATGCATAGGAGCGAGAGTCAAATAGG

387 AAGTCTGGGTAGAAAATAATACAAGGATCAG

388 GATCGCAGGAAGGAACCAACGCAGCTTTGGG

389 ATAGACCTCCCTATAGGAAGAGCGTGGATCG

390 AGGCAATTAGCGCGGCAGGAAATCAAAAAGG

391 TACGTGAAAATCCACGATAAAGCGGAGAGGG

392 GGCAATTTTGTATCTAGTTAACTAGGCCGG

393 ATATAGATACGCCCTCAAATAGGGCATGAAG

394 ACACTCGGTACCAGAGATCAAGTTATGATAG

395 AAAAGAAGGGTCAGACACGCGAAATGCGTTG

396 ATAAAACCGCAGTAGCAAATGCCTGATCTG

397 GGCCGATGGTCAGCTTCCAGGGCTCGCAAGG

398 ATGCACGTTCAGAGTCTTCCCCCCCAGACGG

399 AAGACTCATCAGTAGGGCAGG

400 GGCAAGATTGTTATGCACGGGTAGAG

401 AACCGGTATTACCAATTGACGTAAGGCGG

402 AGATAGCCTCTGGAAACTGCCTGTAGACGG

403 GTGGCCAGCTTAAGAATCCTACAAATTAAAG

404 CGTTACCAAGTACACCAGACAGAAGAATCAG

405 GCCTGTTCAGAGCATACAACCACCAAAATTG

406 ACACATAACCTGAACAATATCCAAAGAAATG

407 CGGCGACCTACGTTGGCACAAAAAGCCTTGG

408 GTGGTAAGAGGCGTGTTAGAGGATAAATGAG

409 GAGAAGAGAATATGGCCTCTAAAGAGTGTAG

410 AACGTTGTAGTGTCATGTCAGTATGAGAGCG

411 CCAGAAACGATCTAATAGAGCTTGCAATTAG

412 CACGGACAAGAGAGACTTTATACCTGCATCG


413 ATCGCTTTTAGGCGCATATTTGCTTTAAAGG

414 GTGCGCTATAAAGGGAACTGGCTTTATAGGG

415 ACAAAACCTTTGCGACGGAATAAGCGGACGG

416 AGACAGACAAAATCAATACGCGAGTTAACTG

417 GCGCGCCATTACTTGAAAGG

418 GGGTCGCACCATATAGGGGTGTAAGGTATAG

419 CCCAGGGAGGCATCAAATTAAGAATACGTGG

420 AGGCGCAACTTATTGACGAAGTCAAAGGGAG

421 ATTAAAAACTGATTCGCTGGATGGCAGAACG

422 GTCGATATCAAGCACGGAACGCTTATCGTAG

423 TAAGGGCAAAGAAGAGCATCCAGAAAGGCTG

424 CCCAAAGTGGTACGGCTTTATAATCCATTTG

425 GATACATAACAGGGAAAGTCGGGGAAATAGG

426 ACTATGAAATCTCTGCCTAGTAGAACGGCGG

427 AACTGAATATCCTTAAGCTGAAAATAAAACG

428 TGGCGGACTAGCTCAGAAATAAAAAACGACG

429 GACGACCCGAAAGTCTCCCACGGACTAACGG

430 AACTGCGCAGTTCAGCTATATGAACGAGCAG

431 ATAATCCATTTGAGCTTCAGCACCCTTGTGG

432 TAAACAGGATATATGGAATCTGCTGTTGATG

433 AGCAATATAAGCGGCAGGCCAAAG

434 GAACTTTTTGGCTGGATAAGCTATGGACGCG

435 TGAGACCACGACGAGCTGCGGGCACAAG

436 GAGGTGGCGGACAGGTTAGGGGTATACTCGG

437 AACACTAAATAAAGTTTATGTGAACCTTATG

438 GCTTATTCGACTCTAACCCAGTGGG

439 CACAGAATTTAACGACTAGGGGCCCGTTAAG

440 GTGGAAGCAAACGCATCCAACTTATGTGATG

441 ATTCGGTACGAGACACGCATACGACGACTAG

442 GTCGAAGGAACTGCAACTAGAGCACAGTCG

443 TAAGGGAACAGAAGCCAGGTGGGGAGGATCG

444 GAGGACCACGTTTGGGAACAACGGCGGATGG

445 CAAGTTCACATATCGTTCGGTAAGAGAACAG

446 TAAGTGAGATGAAGACGTACTTAGAAAATG

447 TGGTTTCCTCCAAGTCGGATTTCGAGTCGCG

448 TAATAAACAACGAAACATAGCTGTGACGCGG

449 TCCGGGAATGAGCGTAGTATTAAACCTCACG

450 CGATGCGGGAATTGGTTTACATGATATTATG

451 CGTCATGCTAGCAAGGTGACACCTCGGTGAG

452 AAAGAGCAGTCCGGTACTTGAGGTCAGGCAG

453 TGCTGAACCATGCCTTGGAAACTTTCTCTAG

454 GGAGGCGAGCACAGCAAATCGTAGCAG

455 TTCGACGACATAGGATGGTGAGTTGAAAATG

456 TAAAACGCAGAAACGCTATGGGACCGTTACG

457 CAAACAGGGGGATAACGGGCTTCATAGGGGG

458 GGATTGTGACAAGTGTCTTACTATGTCTCGG

459 AAAGGTATTATCATTAATAGGATGCCCTGCG

460 ATGACTGTGAGGACAATACAAGGCAAGAGAG

461 TGCGAGAGCTAGTGATGTGTTAAAAGTCGCG

462 ATCTTGACCAGCCGGAGTATCGTAGGGAGTG

463 GAACGCCCAACAATAGATCGGATAGATCGAG

464 CAACTTTACAGGCACGAGTATCACGGAGGAG

465 TGGCGACGACACCCGTTTCGATCTTGCTTTG

466 AGGCAGATACAGGATTAGCCTTATTAGCAAG

467 TATGGGCTAGCGTTACAGGAAGAGGACCGG

468 AAATCCGCTAAGGCATGACCATGACGGTTTG

469 TGTATTCCTTATTCGACTGGGTAGCGACTCG

470 TAACCTAATAGAGATCCACTAGTGTGTAGGG

471 GACGTTAAGAAGCTCCTCTCCACAGGCAAGG


472 AAGAAATCCTTGGTGACGGAACGTTGAATTG

473 GACCGCGATCTCTACAAAGCGGGTTAAAAGG

474 TTGTGGTGAGTAATAGGGTGCAAGGGGAGGG

475 CGGGTCCAGGGTTAGCCCACCTGTTTAGTAG

476 TGACTGCTGCGAACAATGAAATAATGGCAAG

477 GGCGCAGCAAAAATACGGGTATACAGGCATG

478 GACGAAGCGGAGGTGACTGTTCACATTCGGG

479 GACGGTCCAAATAAATAACCTTATCCGATGG

480 GTATTAGGAGGGGACTGGGAGAAAGAAACAG

481 CATAGTTACTTAGCGGGATATACGGTG

482 ACGGGACTCTCCTGACAAACCACACAATGCG

483 GGCGCTAGTTATGATGTGGGGATACACTAGG

484 ACTAGCGGCACCGAAGCGCGCTCGATATCAG

485 ACAAATCTTCATATTCGGCTGACGCTAAGCG

486 CAATAAATCCGGATCATCCACAGTCCGCTGG

487 CACGTAGAAGCGCTTGGAGCGATCAGGCATG

488 AAGGTTCTGGTTAGGTGAAATGTATGAAGAG

489 CATCTTCATGTCGAACAAGTCCGTATG

490 TGAGGAGTGATGTTAGAGTTGAAGG

491 TCCCTATAGCCCTCTAATAAAGTCGATAAGG

492 CAGTAGGTAAAAAGTTCAACGGAAACAACAG

493 TACCATGCAAGGAGCTATGCGCGGTACAG

494 GCTTGGTTAGGGGTGCAAATCAAGCTGAAGG

495 GTGTCGTCCTTGCTCCTATCTTGGAGGAAAG

496 GCCTGTTGAAGGTAGCGCCAAATGGAATGAG

497 AAATCCCCGGTTCTGATCATGTCAG

498 TTAAAATAAACCCAAGGTCGATGGATAGACG

499 CTATCAAACAAAAGAAAGAATACTCCGGTCG


Documents

Nature Methods: doi:10.1038/nmeth...barcode. Bottlenecks were simulat ed by multinomial sampling with replacement and we used equations (1) and (2) from Krimbas & Tsakas 5 to determine