Computational protocol: Plasmodium falciparum genetic crosses in a humanized mouse model

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Protocol publication

[…] Two µg of DNA was sheared using a Covaris S-series sonicator (Covaris; duty cycle 20%, time 180 s, intensity 5, cycle burst 200, power 37 W, temperature 7 °C, mode freq sweeping) from the two parents (NF54HT-GFP–luc×NHP*) and 14 recombinant progeny from the cross. Sheared DNA was end-repaired, A-tailed and multiplex-indexed adaptors ligated using NEBnext library preparation kits for Illumina (New England Biolabs). We replaced the DNA polymerase with Kapa HiFi (Kapa Biosystems) , and used Agencourt AMPure XP beads (Beckman Coulter) for sample purification. The Kapa SYBR Fast ABI Prism qPCR kit (Kapa Biosystems) was used to quantify templates before multiplexing (13 samples/lane) and sequenced on an Illumina HiSeq 2500. Raw Sequence data was de-multiplexed and .fastq files generated using CASAVA 3.0 before further analysis.101 bp paired-end reads from .fastq files were mapped against the P. falciparum genome reference strain 3D7 v9.2 (http://plasmodb.org/common/downloads/release-9.2/Pfalciparum3D7/fasta/data/) using BWA v0.6.1 . The resulting BAM files were cleaned to remove reads which map off chromosomes and PCR duplicates removed using picard v1.56 (http://picard.sourceforge.net/). The Genome Analysis Toolkit v2.3-9 was used to realign around indels and generate/re-calibrate base quality scores before final SNP calling was performed using the UnifiedGenotyper. Variant quality scores were then recalibrated and variants removed if they failed any of the following quality metrics (QUAL < 100.0, FS < 50, BaseQRankSum -2 > X > 2, MQRankSum -2 > X > 2, QD < 10).A genetic map was constructed using the est.map function in R/qtl in R v3.1.0 using the Haldane map function. As the parental lines were sequenced directly each SNP in the progeny was designated by its parent of origin. We calculated pair-wise allele sharing between progeny using the comparegeno function, and assessed if the observed data conformed to a normal distribution using the Shapiro-Wilk test for normality, implemented in the shapiro.test function in R. […]

Pipeline specifications

Software tools BaseSpace, BWA, Picard, GATK, R/qtl
Databases PlasmoDB
Application WGS analysis
Organisms Mus musculus, Plasmodium falciparum, Homo sapiens, Pan troglodytes
Diseases Malaria