Supporting InformationBozdech et al. 10.1073/pnas.0807404105SI TextSample Collection and RNA Isolation. Adult patients from North-western Thailand (Shoklo Malaria Research Unit, Mahidol Uni-versity, Thailand) with symptomatic vivax malaria gave informedconsent to donate 10 ml of blood. The P. vivax infected bloodsamples were collected from three preselected patients beforereceiving standard chloroquine treatment. These samples con-tained �1% of RBCs infected with synchronous P. vivax populationat ring stages estimated as �9 h after invasion (1). Each of the P.vivax isolates was genotyped to determine its clonal status asdescribed (2). CF11 columns were used to minimize the presenceof host WBCs, and the parasites were cultured as described (3).Briefly, the P. vivax infected blood cells were cultured in a 2%hematocrit blood media mixture consisting of McCoy’s 5A and 20%AB� human serum. The total volume of blood media mixture foreach isolate (90mls) was split evenly into nine culture flasks, eachrepresenting an individual time point (time point, TP1–9). Exceptfor the first time point (TP1), each culture flask was charged witha gas mixture of 5% CO2, 5% O2, and 90% N2 for 1min and thenplaced into a 37.5°C incubator. Cell samples were collected forRNA isolation immediately after adding to the flask (TP1) and thensubsequently at 6-h intervals (TP2–9). Total RNA was isolated byusing a standard protocol of trizol/chloroform extraction as de-scribed (4).

Target DNA Preparations and Microarray Analysis. For preparationof the target DNA for microarray hybridization, we adapted theSwitch Mechanism at the 5� end of Reverse Transcription(SMART) PCR approach as described (5). Briefly, 1 �g of totalRNA and a mixture of three oligonucletotides (AAGCAGTG-GTATCAACGCAGAGTACGC-(T)30, AAGCAGTGGTAT-CAACGCAGAGT (N)9, and DNA:RNA oligonucleotide hy-brid d(AAG CAG TGG TAT CAA CGC AGA GTACGC)r(GGG) at concentration 1:1:2 �M was resuspended in 10�l of water, denatured at 70°C for 10 min, and subsequentlyplaced on ice for 10 min for annealing. Thereafter, reversetranscription (RT) was carried out by using the reverse tran-scriptase with intrinsic cytosine-specific terminal transferaseactivity (PowerScript, Clontech) for 2 h at 42°C at a final volumeof 20 �l. Subsequently 1/10 of the RT reaction mixture (equiv-alent to 100 ng of starting RNA material) was directly introducedinto the PCR mixture that consists of standard Tag Polymerase(NEB) (5U/�l), primer (AAGCAGTGGTATCAACGCA-GAGTACGC) (6 �M), dNTPs (ATP:CTP:GTP:TTP: aaUTP

�2:1:1:1:1) (total dNTP concentration 10 �M) and a reactionbuffer at a final volume of 100 �l. The thermocycling was carriedout as follows: 95°C for 5 min, 18 cycles at 95°C for 30 s, 60°Cfor 30 s, and 68°C for 5 min. The resulting PCR product waspurified by using the MiniElute DNA purification kit (Qiagen).The DNA was labeled with the fluorescent dyes (Cy5 and Cy3),and the microarray hybridization was carried at 65°C in theautomated hybridization station (Maui, USA). The microarrayanalyses were performed as two channel competitive hybridiza-tions where RNA from each time point was labeled by Cy5 andhybridized against a reference RNA pool labeled with Cy3. Thereference RNA pool was assembled by combining equal mass oftotal RNA from all nine time points from all three P. vivaxisolates.

Data Processing and Analysis. Microarray data processing andanalysis (including the Fast Fourier Transform) was carried outas described (6). The overview phaseogram (Fig. 1) containsgenes with �2-fold change in mRNA abundance across the IDC,and their expression profile exhibit �40% of total power in themain component of the power spectra. For the P. falciparumtranscriptome comparisons, we used the quality controlled,high-density IDC transcriptome for the HB3 strain obtainedfrom the P. falciparum transcriptome database (http://malaria.ucsf.edu). For the analyses of differential gene expres-sion, we calculated the total sum of the original expression ratios:

A � �i�1

g

expTPi

Where expTPi is the original expression ratio measured by themicroarray analysis for each time point (i). Subsequently, wecarry out pair-wise analyses of the A values between the P. vivaxisolates to evaluate the differential mRNA abundance for eachgene for all possible isolate pairs as follows: The differential inmRNA abundance is calculated as dAj.k-l � log2(Aj,k/Aj,l) wherej corresponds to P. vivax gene; k and l is any given isolates from(smru1, smru2, and smru3) where k � l. For each of the isolatepair, we calculate the median (�k-l) and standard deviation (�k-l)of the dAk-l distribution. For the purpose of these studies, geneswith dAk-l outside of the interval (�k-l � 2* �k-l, �k-l-2* �k-l) (Pvalue � 0.05 for normal distribution) are classified as differen-tially expressed.

1. Chotivanich K, et al. (2001) Ex-vivo short-term culture and developmental assessmentof Plasmodium vivax. Trans R Soc Trop Med Hyg 95:677–680.

2. Imwong M, et al. (2007) Relapses of Plasmodium vivax infection usually result fromactivation of heterologous hypnozoites. J Infect Dis 195:927–933.

3. Russell BM, et al. (2003) Simple in vitro assay for determining the sensitivity ofPlasmodium vivax isolates from fresh human blood to antimalarials in areas where P.vivax is endemic. Antimicrob Agents Chemother 47:170–173.

4. Bozdech Z, et al. (2003) Expression profiling of the schizont and trophozoite stages ofPlasmodium falciparum with a long-oligonucleotide microarray. Genome Biol 4:R9.

5. Petalidis L, et al. (2003) Global amplification of mRNA by template-switching PCR:Linearity and application to microarray analysis. Nucleic Acids Res 31:e142.

6. Bozdech Z, et al. (2003) The transcriptome of the intraerythrocytic developmental cycleof Plasmodium falciparum. PLoS Biol 1:E5.

7. Carlton J (2003) The Plasmodium vivax genome sequencing project. Trends Parasitol19:227–231.

8. del Portillo HA, et al. (2001) A superfamily of variant genes encoded in the subtelomericregion of Plasmodium vivax. Nature 410:839–842.

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Fig. S1. Correlations of individual transcriptome datasets. Histogram showing the distribution of Pearson correlation coefficients (PCCs) calculated geneexpression profiles determined by the microarray expression analyses: (A) SMRU1 and its replicated dye-swap experiment. (B) Isolates SMRU1 and SMRU2. (C)Isolates SMRU1 and SMRU3. (D) Isolates SMRU2 and SMRU3. (E and F) Histograms of PCC values of the expression profiles between SMRU2 and the P. falciparumHB3 (E)and SMRU3 and the P. falciparum HB3 (F) are shown. The calculations include genes with expression profiles that show �2-fold difference between themaximum and minimum value across the IDC. In the smru1 dye swap experiment, �98.3% of expression profiles show PCC �0.7, and in the biological replicasof different strains, �85% of genes show PCC �0.7. (G) Verification of microarray results by quantitative RT- PCR of five selected genes. The log2-transformed,mean-centered expression ratios derived from the microarray hybridizations of SMRU1 and replicate dye-swaps were plotted for five genes along the time course(Time Point 1 thorough 9 on the x axis) (‘‘Microarray’’ and ‘‘Dye swap’’). mRNA abundance for the five genes were determined as �Ct by using real-timequantitative RT-PCR. The RT-PCR results were normalized to the reference pool (identical to the microarray experiment) and plotted along the microarray data.Each reaction was carried out in triplicate, and the average mean-centered �Ct values together with the standard deviations were determined and plotted foreach time point. There is complete agreement with the gene expression profiles analyzed by both methods.

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Fig. S2. Hierarchical clustrogram of the IDC gene expression profiles of vir gene family. Of the 346 vir genes predicted in the P. vivax genome, the microarraycontains unique oligonucleotide probes for 262, and of these, 204 were found to be expressed in the P. vivax IDC. The heat map shows distribution of the virgene expression expressed as log2 transformed, mean-centered mRNA abundance subjected to hierarchical clustering that recognize several major clades inwhich the vir gene expression is highly correlated. There are �30 vir genes expressed in the late schizonts and less so in the early rings, 18 vir genes expressedin the early schizonts, and 2 small groups (clades) of vir genes expressed somewhat earlier (4 clades from the top). Furthermore, �100 vir genes were found tobe expressed exclusively in the ring stage, and 25 virs expressed in the late ring and early trophozoite. The colored bar (on the right) indicates the phylogeneticgroup of vir genes defined by the P. vivax genome sequencing project (7, 8).

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Fig. S3. Gene expression of P. vivax gene families. Expression profiles of 10 merozoite surface proteins (MSP3), 11 MSP7, 9 reticulocyte binding proteins (RBP),21 PHIST, and 34 RAD genes is shown. TheRAD and PHIST gene families are evenly divided between schizont specific transcripts (11 PHIST and 17 RAD) andring/trohozoite transcripts (10 PHIST and 17 RAD). The entire MSP3 and RBP families and the majority of the MSP7 gene family are expressed in schizonts. Theheat map represents the individual gene expression profiles that were log2 transformed and mean centered.

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Fig. S4. Correlation analyses between P. falciparum and P. vivax transcriptomes. The plots depict the array-to-array PCCs between 9 time points of P. vivax and46 time points of the P. falciparum (HB3 strain) transcriptome. Each function (curve) represents the profile of PCCs between a particular P. vivax IDC transcriptometime point and all time points in the from the P. falciparum IDC transcriptome. The PCCs were calculated in array-to-array fashin including all syntenic genes withexisting expression profiles (2,426) (Fig. 2) in both P. vivax and P. falciparum transcriptomes. For the subsequent gene based Pearson correlation analyses, eachP. vivax transcriptome time point was matched to the P. falciparum transcriptome time point in which the PCC reached the highest value (arrow).

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Fig. S5. Correlations between IDC expression profiles for P. falciparum–P. vivax gene pairs within functional groups. Distribution of Pearson correlation valueswithin the gene functional groups described by the Malaria Parasite Metabolic Pathway database (http://sites.huji.ac.il/malaria/). The 80th and 90th percentileof the Pearson correlation value distribution within each functional group is represented by the colored boxes and ‘‘error bars’’, respectively. The median Pearsonvalue is indicated by the black line within each 80th percentile distribution box. The black circles indicate the 95th percentile outliers. The x axis labels indicatethe functional group, median Pearson correlation, and number of genes within the functional group considered for this analysis.

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Table S1. Monitoring of the parasites culture synchronicity bycounting of morphologic stages

Timepoint

Hours in exvivo culture Rings, %* Trohozoites, %* Schizonts, %*

smru1† (parasite count 10/1000 RBC �1.0% overall parasitemia)1 0 100 0 02 6 n.d. n.d. n.d.3 12 n.d. n.d. n.d.4 18 0 100 05 24 0 100 06 30 0 76 247 36 0 18 828 42 0 40 609 48 0 0 30smru2† (parasite count 7/1000 RBC �0.7% overall parasitemia)1 0 83 17 02 6 37 57 63 12 10 85 54 18 5 94 15 24 15 83 26 30 7 81 127 36 16 39 458 42 6 29 659 48 30 53 17

n.d. not determined.*Giemsa stained smears from each time point culture samples were visuallyinspected and representation of each morphological stage was counted.

†The stage count was determined for two of the three P. vivax isolates: smru1used for the P. vivax IDC transcriptome overview dataset (Fig. 1) and smru2.

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Other Supporting Information Files

Dataset S1 (XLS)Dataset S2 (XLS)Dataset S3 (XLS)

Table S2. Representatives of functionally significant genes with altered IDC transcriptional regulation between P. falciparum andP. vivax

Functional groupP. falciparum

geneP. vivaxGene

Pearsoncorrelation

P. falciparumexpression*

P. vivaxexpression* gene annotation

genes coding fortransport proteins

PFF1430c Pv114575 0.0362 trophozoites early schizont Transmembrane amino acidtransporter

MAL13P1.271 Pv114705 0.286171707 early rings early schizont V-type ATPase, putativePF13_0034 Pv122165 0.0278 early rings early schizont Vacuolar ATP synthase subunit HPF08_0113 Pv088205 0.0022 early rings early schizont V-type ATPase subunit A

hemoglobindegradation

PF14_0075 Pv086040 0.155693152 early rings early schizont Plasmepsin IVPF14_0076 absent na early rings absent Plasmepsin IIPF14_0077 absent na late rings absent Plasmepsin IPF14_0078 absent na late rings absent Plasmepsin III (HAP)PF11_0161 Pv091410 0.074698552 early rings late rings falcipain-2/vivapain3

parasitophorous vacuoleand Maurer’s cleftsproteins

PFB0100c Pv003520 0.7316 late rings late schizont KAHRPPF14_0678 Pv116915 0.2992 late rings late schizont Exported protein 2, Exp 2PF11_0224 Pv091700 0.6169 early rings late schizont Circumsporozoite-related antigen;

Exp-1PF08_0036 Pv089235 0.3314 early rings early schizont Transport protein; PfSec23APFB0640c Pv002830 0.6696 early rings early schizont Hypothetical protein; PfSec31pPFD0310w Pv000930 0.6149 early rings early schizont Sexual stage specific protein

precursorPF10_0164 Pv001715 0.1993 early schizont late schizont etramp10.3PF10_0323 Pv111065 0.6375 late rings early schizont etramp 10.2PF13_0012 Pv121950 0.9390† early rings late schizont etramp 13PFB0120w Pv003565 0.6849† early rings late schizont etramp 11.2

protein kinase codinggenes

PF14_0605 Pv117280 0.4566 early rings trophozoites cyclin/CMGCPF08_0044 Pv089305 0.3173 early rings late schizont protein kinase/CMGCPFD0865c Pv089980 0.4330 early schizont late schizont cdc2-related protein kinase 1/CGMCPFC0105w Pv119250 0.0446 early rings early schizont ser/thr protein kinase/CMGCPFF0260w Pv113435 0.2498 early schizont late schizont ser/thr protein kinase/NimAPFL0080c Pv084105 0.3433 early schizont late schizont ser/thr protein kinase Nek1/NimAPF11_0239 Pv091755 0.4688 early rings early schizont Ca-dep protein kinase/CamKPF14_0227 Pv085300 0.0377 late rings late schizont Ca-dep protein kinase/CamKPF11_0242 Pv091770 0.0877 early rings early schizont myosin light chain kinase/CamKPF14_0264 Pv085120 0.1718 early rings early schizont peptide chain release factor 1PF14_0423 Pv118270 0.0309 early rings late schizont ser/thr protein kinase

genes coding forenzymes/proteinsinvolved in DNAreplication

PFF1150w Pv114310 0.167724622 trophozoites late schizont Ribonuclease H1PF11_0117 Pv091205 0.1202 trophozoites late schizont Replication factor-CPF14_0602 Pv117295 0.0398 trophozoites late schizont DNA polymerase (Pola)MAL13P1.134 Pv116650 0.2066 early schizont late schizont HelicasePF10_0114 Pv094805 0.0786 early rings early schizont DNA repair protein RAD23PFI0235w Pv098685 0.0702 trophozoites late schizont Replication factor A-related proteinPFL2440w Pv101435 0.0491 trophozoites late schizont DNA repair protein rhp16PF14_0081 Pv086025 0.2199 trophozoites early schizont DNA repair helicasePFA0390w Pv081385 0.5689 trophozoites late schizont DNA repair exonuclease

pre-replicative complex PFB0720c Pv002750 0.3504 early schizont late schizont ORC5PF13_0189 Pv082635 0.2581 early schizont late schizont ORC4PFL0150w Pv084195 0.1922 early schizont late schizont Cdc6p

Abbreviations: KAHRP, knob associated histidine rich protein; CMGC, cyclin-dependent, mitogen-activated, glycogen-synthase, CDK-like protein kinase class;NIMA, never-in-mitosis/Aspergillus protein kinase class; CamK, Ca-dependent kinase class; HAP, histo-aspartic protease.*Corresponds to peak expression within each IDC transcriptome.†High person values are because of the periodic character of the P. falciparum expression profiles.

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