Population Biology Correction for “Evidence of strain structure in Plasmodium falciparum var gene repertoires in children from Gabon, West Africa,” by Karen P. Day, Yael Artzy-Randrup, Kathryn E. Tiedje, Virginie Rougeron, Donald S. Chen, Thomas S. Rask, Mary M. Rorick, Florence Migot-Nabias, Philippe Deloron, Adrian J. F. Luty, and Mercedes Pascual, which was first published May 1, 2017; 10.1073/pnas.1613018114 (Proc. Natl. Acad. Sci. U.S.A. 114, E4103–E4111).
In the “PCR Amplification for var DBLα Typing” section of the Materials and Methods, the authors note that incorrect primers and conditions used to generate the data were reported. The authors state: “The mistake arose due to change in personnel, leading to reporting primer sequences used in an earlier version of the method. The correct method was published in Rask et al. (2016) and was used for the generation of data reported in this PNAS paper. The error in reporting the incorrect primer sequences means that a longer PCR product would have been generated. The impact of this error is that the previously reported primers would not work for platforms for short read sequences such as Illumina.”
The “PCR Amplification for var DBLα Typing” section of the Materials and Methods, beginning on page E4108, right column, fourth paragraph, line 1, should instead appear as:
From the genomic DNA, we performed PCR amplification of the DBLα domain of the var genes using fusion primers for multiplexed 454 Titanium amplicon sequencing as previously described (59). The DBLα domain has been used previously as a marker of var gene diversity in other investigations (6–8). We coupled template-specific degenerate primer sequences targeting homology block 2 and 3 (5, 14): DBLαAF, 5′-GCACGMAGTTTYGC-3′ and DBLαBR, 5′-GCCCATTCSTCGAACCA-3′. Each of the forward and reverse DBLα primers was barcoded with a 10 bp multiplex identifier (MID) tag published by Roche (Roche 454 Sequencing Technical Bulletin No. 013-2009; 454 Sequencing Technical Bulletin No. 005-2009). Intervening primer sequence necessary for the 454 Titanium platform was included in these fusion primers. These primers were validated for amplification of sequences of the appropriate length using P. falciparum 3D7 genomic DNA (59).
All PCR reactions for the DBLα amplification were carried out in a total volume of 40 μL composed of 3 μL of each primer (10 μM), 1.4 μL dNTP mix (2 mM), 4 μL buffer 5×, 2 μL MgCl2, 0.6 μL Taq polymerase (Promega, GoTaq polymerase, 5 UI/μL), and 2 μL of isolate genomic DNA. PCR cycling was carried out on an Eppendorf EP Gradient Mastercycler and involved 95 °C for 2 min, followed by 30 cycles of 95 °C for 40 s, 49 °C for 1 min 30 s, 65 °C for 1 min 30 s, and a final extension step of 65 °C for 10 min. PCR amplification was confirmed visually by gel electrophoresis (2% agarose in 0.5× TBE buffer) with nucleic acid staining (EZ VISION™ DNA Dye, Ambresco) demonstrating a band of the appropriate size (~477 bp). Positive controls (laboratory genomic P. falciparum DNA) and negative controls (no template) were performed for quality assurance. The PCR products were then purified using solid-phase reversible immobilization method (Agencourt, AMPure XP). Then, PCR amplicon concentrations were measured using the Quant-iT PicoGreen dsDNA kit per the manufacturer’s instructions (Invitrogen). Known concentrations of control DNA were prepared as directed by the Roche Technical Bulletin (454 Sequencing Technical Bulletin No. 005-2009). We assayed fluorescence intensity using a Perkin-Elmer VICTOR X3 multilabel plate reader, with fluorescein excitation wavelength of ~480 nm and emission of ~520 nm wavelength. We prepared PCR amplicon library pools, each containing equimolar amounts of the PCR amplicons with unique MID tags.
These pools were sequenced in forward and reverse directions on segregated regions using 454 GS FLX Titanium chemistry (Roche). Sequencing was performed by Seqwright Genomics (Houston, TX, USA) and New York University Genome Technology Center (New York, NY, USA).
As a result of this change, the authors note that two references should be removed:
59. J. D. Smith, G. Subramanian, B. Gamain, D. I. Baruch, L. H. Miller, Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family. Mol. Biochem. Parasitol. 110, 293–310 (2000).
60. M. Hamady, J. J. Walker, J. K. Harris, N. J. Gold, R. Knight, Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nat. Methods 5, 235–237 (2008).
Authors also note that reference 62 has been moved to reference 59 as a result of the changes described above.
These corrections do not affect the conclusions of the article. The online version has been corrected.