Molecular barcode diversity from Dielmo and Ndiop, Senegal. A,
Barcodes from samples included in this study. Blinded sample codes, Patient
parameters are shown including sample ID (color coded per region of collection),
date of collection, sex, age, parasite density /µL, and collection location.
Single-nucleotide polymorphism (SNP) calls are shown for each of 24 markers and a
call of “N” represents a mixed-allele call. M/P genomic indicates monogenomic or
polygenomic infections, based on number of Ns; ≥2 represents a polygenomic
infection. Based on the barcodes, complexity of infection (COI) is also shown as
estimated by the COIL algorithm. Common genetic signature (CGS) cluster haplotypes
are listed and colored on the far right. B, Polymerase chain
reaction (PCR) sequencing for marker A1 reveals a novel polymorphism in the
probe-binding region for sample IP20. PCR sequencing to confirm SNP assay A1 call
was performed with the same primers used for the high-resolution melting amplicon:
A1 F: 5ʹ-AGCAATCGAACCCTTTGATCTA-3ʹ; A1 R: 5ʹ- AATGAGAAGAGAGAAATATGTGTTGATAA- 3ʹ and
the same PCR conditions, as described previously [7]. PCR products from sample and controls (3D7 and Dd2) were
run on a gel to confirm size and purity and were sequenced by Sanger sequencing in
both forward and reverse directions using the same primers. Contiguous sequences
were generated and alignments were performed using MacVector (version 12.7.3).
C, Neighbor-joining phylogenetic tree of molecular barcodes using
the Jukes–Cantor method. Sample names at the branch ends are colored per sample
location. CGS haplotype clusters are indicated by colored bars. D,
Map of sample locations and travel history for individuals with the haplotype 3
parasite genotype. Base map of Senegal (left) is based on United Nations map of
Senegal (map number 4174, revision 3, January 2004); zoomed in panel (right) was
created with TileMill Version 0.10.1 (MapBox) with terrain data from Natural Earth.
Legend indicates major cities and villages of interest (diamonds: Dielmo, Ndiop,
Thiès, Kaolack, Kaffrine), and color-coded travel histories for the 6 individuals
with the haplotype 3 parasite genotype. The size of the circle indicates the number
of trips to each location.