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Pipeline publication

[…] b'ion kits for Illumina (New England Biolabs). Following (; ) 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 they were multiplexed (12 samples/lane) and sequenced on an Illumina HiSeq 2000. Raw Sequence data were demultiplexed and. fastq files generated using CASAVA 3.0 before further analysis., One hundred one base pairs 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 polymerase chain reaction 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/recalibrate 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 \xe2\x88\x922>X>2, MQRankSum \xe2\x88\x922>X> 2, QD < 10). For P. falciparum analysis well annotated SNPs were used for variant quality score recalibration (http://plasmodb.org/plasmo/). For nonpooled samples we additionally recalled SNPs using the GATK HaplotypeCaller from v2.8-1 of the Genome Analysis Toolkit after restricting calling to genome regions with sign' […]

Pipeline specifications

Software tools BWA, Picard, GATK