Sequencing and Analysis of Pfcrt Gene

The MID tagged Pfcrt (PF3D7_0709000) primers failed to generate nested PCR amplicons; therefore, Pfcrt was amplified and sequenced using previously described primers and PCR conditions (31 (link), 32 (link)) on an ABI3730xl system (Applied Biosystems). The Pfcrt sequence assembly was performed in CLC Main Workbench v7.9.1 (Qiagen, UK), and SNPs were identified and called based on the 3D7 reference sequences. Nucleotide positions that displayed a peak within a peak in the sequence chromatograms were noted as “mixed.” Consensus sequences were extracted from the sequence assemblies using CLC Genomics Workbench v9.5.3 and used to construct multiple-sequence alignments in Clustal Omega v1.2.1 (67 (link), 68 (link)) to identify SNPs.

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Osoti V., Akinyi M., Wamae K., Kimenyi K.M., de Laurent Z., Ndwiga L., Gichuki P., Okoyo C., Kepha S., Mwandawiro C., Kandie R., Bejon P., Snow R.W, & Ochola-Oyier L.I. (2022). Targeted Amplicon Deep Sequencing for Monitoring Antimalarial Resistance Markers in Western Kenya. Antimicrobial Agents and Chemotherapy, 66(4), e01945-21.

Publication 2022

ABI3730xl system CLC Main Workbench v7.9.1

Based sequences Consensus sequences Nested pcr Nucleotide Primers Sequence alignments Snps

Corresponding Organization : Kenya Medical Research Institute

Other organizations : Institute of Primate Research, National Museums of Kenya, University of Nairobi, University of Oxford

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Variable analysis

independent variables

Primers used for amplification and sequencing of Pfcrt gene

dependent variables

Presence of nested PCR amplicons for Pfcrt gene
Pfcrt gene sequence assembly and SNP identification

control variables

Previously described primers and PCR conditions used for Pfcrt amplification and sequencing
3D7 reference sequences used for SNP identification

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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