Similar protocols

Protocol publication

[…] parasite age was estimated by finding the best fit Spearman Rank Correlation value to a reference transcriptome. VSAs were removed and analysed separately prior to identification of differentially expressed genes. The SAM (statistical analysis of microarrays) approach for time-course data with all 924 possible permutation and FDR cut-off of less than 5% was used., Two micrograms of genomic DNA from each of the clones was sequenced using Illumina Mi-seq sequencing technology with 250 bp paired-end reads. The sequence data were aligned to the P. falciparum 3D7 genome (PlasmoDB version 9.1) and reads that did not map to the reference genome and/or PCR duplicates were removed using Samtool and Picard. The reads were realigned around indels using GATK RealignerTargetCreator and base quality scores were recalibrated using GATK Table-Recalibration. Identification of all possible SNPs in clones was carried out using GATK UnifiedGenotyper (Min Base quality score ≥ 20) by calling variants in the sequence data. This was followed by using GATK VariantFiltration for hard-filtering based on quality scores, read depth etc. to obtain good quality SNPs. SNPs that failed any of these cut-off filters were removed – variant quality as function of depth QD < 2.0, strand bias (P < 10e-6), mapping quality < 40, depth of read < 5, MappingQualityRankSum < −12.5, ReadPosRankSum < −8.0. Genetic variants were then annotated using snpEFF., Four polyclonal antibodies were raised in parallel in mice against the hydrophilic amino acid sequence KNHTNKFHKRKKE, between positions 101th to 113th near the N-terminal of the protein (Biosciences Peptide Synthesis Facility, School of Biological Sciences, Nanyang Technological University). The epitope was selected using the Bcell Parker Hydrophilicity Prediction software., One millilitre of 10% infected pRBC was harvested every 6 h over the 48 h IDC from tightly synchronized in vitro cultures of the clones and lysed with saponin and loading buffer. Ten microlitres of supernatant from the lysed samples were resolved by a 6% polyacrylamide gel and transferred onto the membrane at 4° […]

Pipeline specifications

Software tools Picard, GATK, SnpEff
Organisms Plasmodium falciparum
Diseases Malaria, Malaria
Chemicals Ribonucleotides, Heterocyclic Compounds, 2-Ring, Purine Nucleotides, Heterocyclic Compounds, 2-Ring, Cinchona Alkaloids, Heterocyclic Compounds, 2-Ring, Quinuclidines, Heterocyclic Compounds, 2-Ring