1. Reads aligned into contigs
Barry et al. Figure S1 A.
Step 1: Degenerate primers designed to DBLa conserved amino acid blocks D and H [26]
D H B.
Step 5: Remove vector contamination and edit raw sequence data (“reads”)
Individual reads
Forward primer
Reverse primer
Step 2: PCR of P. falciparum genomic DNA with degenerate primers results in a pool of PCR products containing distinct sequences from different genes
Step 6: Align reads with a 96% DNA sequence identity cutoff
Reads aligned into contigs
Pooled PCR products
Step 3: Ligate PCR products with Topo-TA vector (Invitrogen) and
clone to separate distinct sequences
Step 7: Remove redundancy by generating consensus “sequences” (together they form a “repertoire”)
Repertoire
Colonies
Clones
Sequences
E.coli transformation
Step 8: Alignment of sequences with a 96% DNA sequence identity cutoff defines distinct sequences (“types”) in the population.
Step 4: Culture 96 E. coli colonies in 1ml L-broth plus Kanamycin. Extract plasmid DNA and sequence using M13 Universal primers
Types (1-7) A B C D
96 well plate
Repertoires