doi: 10.1038/nature09352 SUPPLEMENTARY INFORMATION · Supplementary Figure 1. Phylogeny of the...

Supplementary Figure 1. Phylogeny of the populations used in this study. Long periods of independent evolution separate the founding of each experimental population. The ACO treatment imposes a 9/10-day generation cycle and selection for accelerated development. The CO treatment imposes a 28-day generation cycle, with no pressure on development time and moderate selection for postponed reproduction. ACO populations were derived from their same-numbered CO (control) populations in 1991. At the time of resequencing, the CO populations had experienced 252 generations, and the ACO treatments had experienced 605 generations.

SUPPLEMENTARY INFORMATIONdoi: 10.1038/nature09352

www.nature.com/nature 1

Supplementary Figure 2. Depth of sequence coverage. Top two panels depict the number of SNPs (688,520 total) observed at differing sequencing coverage depths for the ACO (top left) and CO (top right) populations. In both populations the average depth is ~20X, but in ACO coverage is more variable. The lower left panel is a similar plot of coverage for the ACO1 library. The lower right panel shows the distribution of minor allele frequencies (MAF) averaged over the ACO and CO libraries. The dearth of low MAFs is due to our quality filtering requirement that a SNP must have a MAF significantly greater than 2% in order to be considered a true positive SNP.

doi: 10.1038/nature09352 SUPPLEMENTARY INFORMATION


Supplementary Figure 3. The proportion of different types of genic events as a function of different minor allele counts (MAC). The genic events consist of nonsynonymous polymorphisms (AA), segregating stop codons (STOP), or segregating splice variants (SPLICE). In the case of nonsynonymous SNPs and segregating stop codons we further characterize if the allele different from the reference genome is associated with the major (mj) or minor (mn) allele of this study.



Supplementary Figure 4. Screen shot from the U.C. Santa Cruz Genome Browser. This screen shot depicts the region surrounding caudal, a homeobox-containing gene involved in gastrulation, morphogenesis, pattern formation, and imaginal disc development.



Supplementary Figure 5. Close-up of a 1.5 Mb region on X. Depicts a subset of the data in Figure 2, but focused on the region from 1.5 Mb to 3.0 Mb on the X chromosome. This shows the typical resolving ability of the experiment.



Supplementary Figure 6: Heterozygosity vs. allele frequency differentiation. Regions with reduced heterozygosity generally show a strong association with the regions strongly differentiated in allele frequency. To illustrate this in the upper panel we have plotted levels of heterozygosity measured in the pooled ACO sample (see the red line in Fig. 3 for reference) against the L10FET5%Q for each window throughout the genome. The lower panel shows allele frequency differentiation plotted against the smaller of the heterozygosity estimates in either ACO or CO pool. Regions showing large allele frequency differences between the ACO and CO pools are generally associated with reduced heterozygosity in at least one of the treatments.



Supplementary Figure 7: Models of selection. The distribution of allele frequencies after 600 generations of selection were obtained by simulating a Wright-Fisher population of N = 1000 diploids, under a model of genic selection (fitnesses 1, 1+s, and 1+2s for the wild-type homozygote, heterozygous mutant, and homozygous mutant genotypes, respectively, with the strength of selection remaining constant each generation). All results are conditioned on the beneficial allele not being lost from the population. For both panels, the neutral expectation shown is for a sample of n = 100 chromosomes sampled from a diploid population at equilibrium for mutation and drift. Top panel: The distribution of allele frequencies for a newly-arisen beneficial mutation with an initial frequency of 1/(2N) based on 5000 replicate simulations. These simulations show that newly arising mutations having selection coefficients >1% are likely to show the signature of a classic sweep, whereas those having selection coefficients of <0.1% are unlikely to display sufficient allele frequency divergence from control populations to be detected in our experiment. Therefore, given the length of the experiment to date, it is possible that classic sweeps may be occurring, but alleles have small associated selection coefficients and have not had time to fix. Bottom panel: The distribution of allele frequencies for a beneficial mutation at an initial frequency of 10% in the population. These simulated predictions of selection from standing variation. Under this soft-sweep model, we see that mutations with selection coefficients >0.5% are likely to fix.



Supplementary Table 1: Functional characterizations of genes where we find nonsynonymous mutations. The results from DAVID, a Gene Ontology analysis of genes harboring nonsynonymous SNP with L10FET scores greater than four.

Term Count % p-

value Genes Pop Hits

Fold Enrich FDR

SP_PIR_KEYWORDS (200) hydrolase 66 13.89% 0.0000 CG11147, CG9323, CG2241, kuz, Ku80, CG3239, CG5776, Pez, Lip2,

CG11874, CG3502, Mbs, CG15385, CG3225, Gli, Hel89B, CG14528, CG11842, kz, CG14869, CG34120, CG11897, CG11898, dob, CG32479, Myo10A, CG3573, CG4049, CG3097, CG9806, CG1494, CG13930, CG3160, Atet, CG5931, CG31213, CG14516, Psn, CAP, CG33080, CG9465, CDase, CG15255, CG9253, CG31233, CG4386, CG15533, CG2493, l(2)35Df, dom, cana, CG10764, Ance-2, CG8562, CG9733, CG1571, eIF-4a, GIIIspla2, CG14526, RluA-1, CG5322, CG11406, spn-B, CG3700, CG10602, CG14529,

1220 3.36 0.00

atp-binding 31 6.53% 0.0000 CG1494, l(2)03659, CG11147, Atet, CG9323, CG5931, CG2241, Klp61F, CG31213, CAP, CG5776, CG9253, lig3, l(2)35Df, hep, l(1)G0020, CG3225, cana, sgg, Aats-pro, kz, CG11897, eIF-4a, CG11898, ik2, ade2, ball, CG2918, Bub1, Doa, Nrk,

607 3.17 0.00

developmental protein 27 5.68% 0.0000 trx, l(2)03659, fliI, trr, crn, tamo, sas, pcx, slou, crb, hep, l(1)G0020, spen, sif, dpp, sgg, ptc, Hrs, H2.0, exu, Idgf5, h, h, Doa, bs, Psc, Nrk,

489 3.43 0.00

transmembrane 37 7.79% 0.0000 Gr23a, Gr58b, CG11147, l(2)03659, CG5687, CG3160, Atet, CG31660, ppk7, FucTD, FucTB, Psn, CG13575, sas, Gr64f, pcx, D2R, Nhe3, Syx4, Prestin, crb, Gr98b, Trc8, tectonic, CG17922, CG12531, tutl, CG9657, ptc, CG11897, ppk14, CG11898, frc, ft, Gr77a, mew, Nrk,

903 2.55 0.00

membrane 37 7.79% 0.0000 Gr23a, Gr58b, CG11147, l(2)03659, CG3160, Atet, CG31660, FucTB, Psn, sas, Gr64f, pcx, Cyp308a1, D2R, Cyp309a1, Nhe3, Syx4, Prestin, crb, Gr98b, Trc8, Cyp4d14, tectonic, CG17922, CG12531, tutl, ptc, CG11897, CG11898, Slh, frc, ft, Gr77a, DLP, mew, Nrk, Cyp309a2,

961 2.39 0.00

metalloprotease 10 2.11% 0.0000 CG3502, CG14528, CG14516, kuz, CG14869, CG3239, CG14526, CG15255, CG10602, CG14529,

78 7.96 0.01

alternative splicing 24 5.05% 0.0000 CG17896, Gr23a, sgg, trx, trr, slgA, CycE, tutl, Psn, per, Hrs, rab3-GAP, sas, sr, Slh, resilin, D2R, Trc8, h, hep, spen, Doa, mew, sif,

496 3.01 0.01

glycoprotein 21 4.42% 0.0000 dpp, Gr58b, l(2)03659, CG3160, FucTB, Psn, ptc, per, CDase, sas, pcx, Idgf5, D2R, ft, Gr77a, Gr98b, crb, tectonic, mew, Nrk, LanB2,

400 3.26 0.01

nucleotide-binding 30 6.32% 0.0000 CG1494, l(2)03659, CG11147, Atet, CG9323, CG5931, CG2241, 767 2.43 0.02



Klp61F, CG31213, CAP, CG5776, CG9253, lig3, hep, l(1)G0020, CG3225, cana, sgg, Aats-pro, kz, CG11897, eIF-4a, CG11898, ik2, ade2, ball, CG2918, Bub1, Doa, Nrk,

protease 16 3.37% 0.0002 CG10764, CG14528, CG11842, CG9733, CG14516, Psn, kuz, CG14869, CG3239, CG14526, CG15255, CG4386, CG3502, CG3700, CG10602, CG14529,

313 3.18 0.23

helicase 7 1.47% 0.0011 CG5931, CG9323, kz, l(2)35Df, eIF-4a, CG3225, CG9253, 73 5.96 1.59 nucleus 25 5.26% 0.0027 Smr, trx, trr, CG5931, crn, l(1)1Bi, pen, fs(1)K10, slou, l(1)G0020, spen,

CycE, per, Nop60B, H2.0, Hr4, h, yemalpha, CG4061, h, ball, Doa, CG9246, Psc, bs,

814 1.91 3.95

aminopeptidase 5 1.05% 0.0028 CG31233, CG3502, CG14516, CG10602, CG9806, 37 8.40 4.01 zinc 20 4.21% 0.0036 trx, CG14528, CG14516, CG11321, CG6613, kuz, CG2662, CG14869,

tamo, CG3239, Hrs, CG14526, CG15255, CG10737, Hr4, CG3502, Trc8, CG10602, Psc, CG14529,

601 2.07 5.17

sodium transport 5 1.05% 0.0053 CG5687, ppk7, Nhe3, CG9657, ppk14, 44 7.06 7.44 INTERPRO (316) IPR014001:DEAD-like helicase, N-terminal

10 2.11% 0.0005 Hel89B, CG5931, CG9323, CG4049, kz, l(2)35Df, dom, eIF-4a, CG3225, CG9253,

82 4.28 0.94

IPR014021:Helicase, superfamily 1 and 2, ATP-binding


84 4.18 1.12

IPR001650:DNA/RNA helicase, C-terminal


85 4.13 1.22

IPR006025:Peptidase M, neutral zinc metallopeptidases, zinc-binding site

10 2.11% 0.0007 CG3502, CG14528, CG14516, kuz, CG14869, CG3239, CG14526, CG15255, CG10602, CG14529,

86 4.09 1.33

Biological Process ALL (256) GO:0007444~imaginal disc development

29 6.11% 0.0001 trr, Psn, spag, nej, Mbs, crb, brk, hep, Gli, spen, dpp, sgg, eys, ptc, ru, eIF-4a, pen, frc, Idgf5, tgy, ft, h, pio, h, sev, DLP, mew, Doa, bs,

435 2.21 0.20

GO:0007224~smoothened signaling pathway

8 1.68% 0.0002 fu, sgg, trr, ptc, nej, DLP, boi, disp, 41 6.46 0.35

GO:0002164~larval development

27 5.68% 0.0003 trr, spag, nej, sas, Mbs, crb, brk, hep, Gli, spen, dpp, sgg, eys, ptc, ru, eIF-4a, pen, frc, Hr4, ft, h, pio, DLP, sev, Doa, mew, bs,

419 2.13 0.63

GO:0035220~wing disc development

17 3.58% 0.0004 dpp, sgg, Psn, ptc, pen, frc, ft, tgy, Mbs, pio, brk, h, DLP, spen, mew, Gli, bs,

204 2.76 0.77



GO:0002117~larval development (sensu Amphibia)

25 5.26% 0.0005 trr, spag, nej, Mbs, crb, brk, hep, Gli, spen, dpp, sgg, eys, ptc, ru, pen, frc, Hr4, ft, h, pio, DLP, sev, Doa, mew, bs,

382 2.17 0.90

GO:0007552~metamorphosis

25 5.26% 0.0005 trr, spag, nej, Mbs, crb, brk, hep, Gli, spen, dpp, sgg, eys, ptc, ru, pen, frc, Hr4, ft, h, pio, DLP, sev, Doa, mew, bs,

382 2.17 0.90

GO:0009887~organ morphogenesis

28 5.89% 0.0005 trr, crn, spag, nej, Mbs, crb, brk, hep, Gli, spen, LanB2, dpp, sgg, eys, ptc, ru, pen, frc, tgy, ft, h, pio, h, sev, DLP, mew, Doa, bs,

455 2.04 0.98

GO:0007560~imaginal disc morphogenesis

24 5.05% 0.0006 sgg, dpp, trr, eys, spag, nej, ptc, ru, pen, frc, ft, h, Mbs, crb, pio, brk, hep, sev, DLP, spen, Gli, mew, Doa, bs,

363 2.19 1.05

GO:0048513~organ development

47 9.89% 0.0016 fliI, trr, crn, Psn, spag, nej, pen, sr, Dap160, Mbs, slou, crb, brk, hep, dom, spen, Gli, CG16719, sif, boi, LanB2, sgg, dpp, enok, mud, CG9555, eys, CycE, ptc, ru, eIF-4a, pen, H2.0, Idgf5, frc, ft, tgy, h, Msp-300, pio, h, DLP, sev, Doa, mew, bs, Nrk,

989 1.57 2.89

GO:0003002~regionalization

22 4.63% 0.0019 sgg, dpp, Psn, ptc, ru, eIF-4a, fu, fs(1)K10, exu, frc, h, orb, brk, h, spn-B, hep, DLP, sev, spen, Gli, Doa, mew,

349 2.09 3.41

GO:0002165~instar larval or pupal development

28 5.89% 0.0021 trr, spag, nej, sas, Mbs, crb, brk, hep, dom, Gli, spen, dpp, sgg, eys, ptc, ru, eIF-4a, pen, frc, Hr4, ft, h, pio, DLP, sev, Doa, mew, bs,

498 1.86 3.73

GO:0006508~proteolysis

37 7.79% 0.0025 CG2241, Psn, CG14516, kuz, CG3239, CG15255, fu, CG31233, CG3502, CG4386, CG9649, CG2493, CG10764, sgg, Ance-2, CG14528, CG8562, CG11842, Sp212, CG9733, kz, CG3099, ptc, CG14869, CG14526, CG31326, frc, CG32523, CG32479, CG18557, CG1632, CG3700, CG10602, CG3097, CG14529, CG9806, CG30288,

739 1.66 4.52

GO:0048731~system development

54 11.37% 0.0026 fliI, spag, kuz, pen, sr, Dap160, Mbs, crb, slou, brk, hep, CG16719, Gli, spen, Ptp52F, sif, boi, dpp, CycE, ptc, ru, H2.0, tgy, ft, h, h, bs, trr, crn, Psn, nej, Trc8, dom, LanB2, sgg, enok, mud, CG9555, eys, tutl, Ank2, eIF-4a, pen, ik2, Idgf5, frc, pyd, Msp-300, pio, sev, DLP, mew, Doa, Nrk,

1210 1.48 4.65

GO:0009791~post-embryonic development

28 5.89% 0.0031 trr, spag, nej, sas, Mbs, crb, brk, hep, dom, Gli, spen, dpp, sgg, eys, ptc, ru, eIF-4a, pen, frc, Hr4, ft, h, pio, DLP, sev, Doa, mew, bs,

513 1.81 5.61

GO:0007389~pattern specification process

22 4.63% 0.0033 sgg, dpp, Psn, ptc, ru, eIF-4a, fu, fs(1)K10, exu, frc, h, orb, brk, h, spn-B, hep, DLP, sev, spen, Gli, Doa, mew,

365 1.99 5.79

GO:0007476~imaginal disc-derived wing morphogenesis

14 2.95% 0.0033 dpp, sgg, ptc, pen, frc, ft, Mbs, pio, brk, DLP, mew, Gli, spen, bs, 182 2.55 5.91

GO:0007472~wing disc morphogenesis

14 2.95% 0.0038 dpp, sgg, ptc, pen, frc, ft, Mbs, pio, brk, DLP, mew, Gli, spen, bs, 185 2.50 6.76

GO:0045165~cell fate commitment

17 3.58% 0.0038 dpp, lin-28, crn, CycE, Psn, nej, ptc, ru, pyd, h, orb, slou, h, spn-B, sev, spen, bs,

252 2.23 6.82



GO:0009653~anatomical structure morphogenesis

45 9.47% 0.0042 Apc, fliI, trr, crn, kuz, spag, nej, pen, fu, Mbs, slou, crb, brk, hep, spen, Gli, sif, Ptp52F, LanB2, sgg, dpp, eys, CycE, Ank2, unc, ptc, ru, pen, ik2, Hr4, frc, ft, pyd, tgy, h, orb, pio, h, spn-B, fs(1)Yb, sev, DLP, Doa, mew, bs,

982 1.52 7.36

GO:0007154~cell communication

67 14.11% 0.0043 Gr23a, Apc, mXr, CG31660, CG6613, kuz, CG13575, fu, sr, Dap160, crb, slou, brk, hep, spen, Gli, sif, boi, dpp, Hel89B, CG18646, CycE, ptc, RhoGAP19D, CG14869, ru, CAP, Hr4, CG3573, h, CG30115, Gr58b, trr, Psn, nej, CG31646, CDase, CG33275, Gr64f, CG10737, CG10700, D2R, stnA, CG5521, Syx4, Gr98b, LanB2, sgg, eys, DLP, per, Hrs, rab3-GAP, Toll-9, CG4733, ik2, frc, pyd, Gr77a, CG8155, sev, DLP, mew, Doa, CG10602, Nrk, disp,

1619 1.37 7.60

GO:0048856~anatomical structure development

61 12.84% 0.0047 Apc, fliI, spag, kuz, pen, fu, sr, Dap160, Mbs, crb, slou, brk, hep, spen, Gli, CG16719, Ptp52F, sif, boi, dpp, CycE, ptc, ru, H2.0, Hr4, ft, tgy, h, orb, h, fs(1)Yb, bs, trr, crn, Psn, nej, Trc8, dom, LanB2, sgg, enok, mud, CG9555, eys, tutl, Ank2, unc, eIF-4a, pen, ik2, Idgf5, frc, pyd, Msp-300, pio, spn-B, sev, DLP, mew, Doa, Nrk,

1448 1.39 8.28

GO:0007220~Notch receptor processing

3 0.63% 0.0052 frc, Psn, kuz, 4 24.82 9.11

GO:0051605~protein maturation via proteolysis

3 0.63% 0.0052 frc, Psn, kuz, 4 24.82 9.11

GO:0051604~protein maturation

3 0.63% 0.0052 frc, Psn, kuz, 4 24.82 9.11



Supplementary Table 2. Details of PCR reaction conditions and restriction enzyme digestions used in the CAPS analysis.

rest

rict

ion

enzy

me

chro

mos

ome

posit

ion

maj

_alle

le

min

_alle

le

LOD

diff

left

_pri

mer

righ

t_pr

imer

left

_pri

mer

_sta

rt

righ

t_pr

imer

_en

d un

cut_

prod

uct_

siz

e

cut_

size

_L

cut_

size

_R

AluI 2L 2232356 A T 3.4664 ATCGTTACACATTTCCACGAAGG ACGAAAAGACGCAAGAGGATG 0 277 278 102 176 AluI 2L 4911536 G T 3.01963 CTAATGAGGCTCAGTTGGTCCAC CATTTGCAGGGATCTGGAGAATA 14 264 251 88 163 AluI 2R 17689593 C T 3.2942 ACTCCCACAAGACATGAAACCGT CTCCTTTCAGGGCAGACCCTC 22 273 252 80 172 DpnII 2L 20809585 G T 3.71129 TTTGAATGATATGTGATGGTCGC TATGCATTTCAGCCACGAACTAA 14 293 280 88 192 DpnII 2R 6113926 C T 3.93378 TCATAAGTTGCCACAATGGACAC AATTAACAACCCCATTTTCGACC 30 299 270 72 198 DpnII 3R 15768682 C T 3.42382 AGCCACTCGGACGTACAAATATC AATGATAATCAAATGCAGGACCC 10 263 254 92 162 DpnII 3R 24762764 T G 3.03732 CCTTCCCTAACCCTCTGCTCTAC AGCAGGACCAGGAAAAGGAAC 5 277 273 97 176 DpnII X 8160168 G T 3.61389 GATGCACAAGGCACCACAAAC ACAGGACGGACTCGGATTTTCT 8 265 258 94 164 HaeIII 2L 5094909 C T 3.38466 TTGTCCGTGGACTGCTGAAAC TCACAAGCAGGTCAGTCTCATTG 11 271 261 91 170 HaeIII 2R 8857292 G C 3.03686 TCACCAGCTTCAGTGTCTCTGTC CTACCGTGTTTATGAGTCGCCC 27 298 272 75 197 HaeIII 3L 2915857 C A 3.06567 CAGTAACATTTCCAGAACGTCCAC AAGGTCGTCTACGAATGGATGC 0 299 300 102 198 HaeIII 3R 24815479 C A 4.34699 CAGATAAACAGGGGCACAAACAC CCCTCTCATTCACTTCACATGCT 12 295 284 90 194 HaeIII X 13631368 G A 3.19598 CAAATTGGTGTCCCTCTTCTTGT CAAAATTGTGGAGAAATCGGAAA 10 268 259 92 167 HhaI 2R 14425984 C G 3.72528 TTTCAAGAACACCATCTTCACGA AGCCTCATAGATTAGGGGCACTC 3 291 289 99 190 HhaI 3R 16512714 C G 4.66264 CAAACTTTTGCACTTAACGAGGC GTGTTTCACCGATTTCCAGATTC 5 261 257 97 160 HhaI 3L 9556844 C T 2.97042 AAGTCCTTACGAATGCTCCTTGG CCCATCCGATATGTAGGTGTCAT 4 257 254 98 156 HhaI X 1871820 A G 4.64536 GGACTAACGACTAACACGCCATC ACAACGAGCGTCATTAACATTTG 5 300 296 97 199 HhaI X 2701714 A C 3.48392 ATCTGCTTGGACTGCAGATTTTC ATAAAAGGAAAGACACAAGGCGG 12 293 282 90 192 MspI 2L 4371013 T C 3.35011 TGAGTGAGTATCTGTGAGTGGGC GCAAAAGGACATACTGCGGATAC 0 275 276 102 174 MspI 2L 20943640 G T 4.16157 AAAGCGTTTGTCCCACTGTCTAC CAAATTCAGAGAAAAGCGTGGTAA 1 254 254 101 153 MspI 3L 16280890 G A 3.36223 GGGATGGCTCTGGCTCTATTAAA AGAGTATCATCTGCTGCGGGAT 13 287 275 89 186 MspI 3R 24878507 T C 3.70825 CCTACCAAACGTAGAGGTAGCCA TCTTGTAGATAATCGGAGCGTTCTC 39 300 262 63 199 RsaI 2L 5970141 C T 3.57037 GTTGAACTCTACCTCACGGCATT ACGCATATAGTCGTAGGTCAGCA 19 285 267 83 184 RsaI 2L 20672481 T C 3.60717 AGTGTGATTACGCTGTTCAACGA CAGGCAGACAGACCGACATAAAT 2 299 298 100 198 RsaI 3R 26248323 A G 4.08469 CTTTCGCAGCAGCTTCTTTTG GGGGAACTCGGTCTCTAGCTG 9 260 252 93 159 RsaI X 2359970 A G 4.2034 CTGCGATTCTCTACCTGTGTCAA ACAGACAGGAAGTGGGCTATTTG 32 283 252 70 182 TaqI 2R 20134765 C G 3.20527 CGTGTAAACAACTTTTATTGCCG GATTAAGATGATGATGATGGGGC 0 263 264 102 162 TaqI 3L 9479891 G T 3.35144 TGACGTTTAACAGCGCAACTG CATATCTGCCAGCAAATCACACT 37 288 252 65 187 TaqI 3R 24993068 C A 3.19078 CTGGTTACCTATGTCTTCACGGG TAATGCTCTTTACTTCGGCCTTG 1 286 286 101 185 TaqI 3L 17394700 T A 3.09395 CCCATTTTGTACCTCCCACAATA CGTACGTGGTGTTGCTGTTTCTA 61 277 217 41 176



Details of gDNA preparation 1 µL of gDNA (approximately 400ng/µL) was aliquoted into each well of a 384 well plate. Each plate included gDNA from each of the 350 experimental females genotyped, as well as negative (no DNA) and positive (DNA from females of the sequenced strain) controls. This DNA was dried down by incubating unsealed plates at 65°C for ~25 minutes. PCR Reactions: 4 µL of PCR cocktail was added to each well. PCR cocktail consisted of 0.5 µL forward primer (1.25 µM), 0.5 µL reverse primer (1.25 µM), 0.4 µL dNTPs (2.5 mM), 0.5uL 10X standard PCR buffer (500mM KCl, 100mM Tris-HCl ph 8.8, 15mM MgCl2, 1% Triton-X), 0.15 µL of Taq polymerase, and 1.95 µL dH20. The thermal cycler profile used was: 98ºC/5 mins; 30 cycles of 98ºC/30sec, 58ºC/30sec, 72ºC/30sec; 72ºC/4 mins; hold at 4ºC. Restriction Enzyme Digests: 3 µL of restriction enzyme cocktail was added directly to each unpurified PCR reaction. Restriction enzyme cocktail consisted of 0.5 µL 10X NEB buffer (variable with enzyme), 0.2 µL 1X BSA, 0.3 µL restriction enzyme (10,000u/mL), and 2 µL dH20. Two restriction enzymes required 0.5 µL of enzyme per reaction: MspI and HhaI. These 7uL restriction enzyme reactions were digested according to the vendor (New England Biolabs) specifications.



doi: 10.1038/nature09352 SUPPLEMENTARY INFORMATION · Supplementary Figure 1. Phylogeny of the...

Documents

Transcript of doi: 10.1038/nature09352 SUPPLEMENTARY INFORMATION · Supplementary Figure 1. Phylogeny of the...