PCR amplification of a py235 gene fragment spanning two contigs, PY05995 and PY03534.

(A) A single PCR product of 392bp was obtained using a forward primer designed on DNA sequences from within the last 200 nucleotides of the 3′ coding sequence of PY05995 and a reverse primer from within the first 200 nucleotides of the 5′ coding sequence of PY03534. (B) Alignment of part of the amino acid sequence translated from the directly sequenced PCR product with the relevant PY05995 and PY03534 amino acid sequences (obtained from the NCBI database). The PCR product sequence aligned perfectly with that of the two contigs and filled a 24 nucleotide gap coding for the eight amino acid residues indicated in red. (C) Confirmation of PY05995 and PY03534 concatenation by RNA-Seq. The two sequences were linked in the RNA-Seq data by 153 read pairs that mapped to the end of PY05995, and the beginning of PY03534. The graphs present the expression in the WT (green) and PY01365-KO (red) parasite lines. The two red diamonds joined by a line indicates the position of the qPCR primers.