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Sample GSM5208268

Query DataSets for GSM5208268

Status

Public on Feb 07, 2022

Title

T20031

Sample type

SRA

Source name

P.falciparum field isolate from patient

Organism

Plasmodium falciparum

Characteristics

isolate: In vivo, Blood stage
treatment: Prior to ACT treatment, 0h

Treatment protocol

In brief, samples were collected from the venous blood of malaria-infected patients at admission to the clinic (Hour 0, 0hr) and 6 hours after their respective treatment (Hour 6, 6hr). Depending on their location, patients were randomly assigned different double or triple Artemisinin Combination Therapy (ACT) as follows: Thailand, Cambodia, Vietnam, and Myanmar - dihydroartemisinin-piperaquine or dihydroartemisinin-piperaquine plus mefloquine; Cambodia - artesunate-mefloquine or dihydroartemisinin-piperaquine plus mefloquine; Laos, Myanmar, Bangladesh - artemether-lumefantrine or artemether-lumefantrine plus amodiaquine.

Growth protocol

All samples used in this study (Except ring 3D7 batch controls) were collected from patients involved in Tracking Resistance to Artemisinin Collaboration 2 (TRAC2), a multi-site study that took place between Aug 7, 2015, and Feb 8, 2018. Following collection, blood was subsequently depleted of plasma and buffy coat. Hour 0 samples were additionally purified from white blood cells (WBC) using CF11 columns following the Worldwide Antimalarial Resistance Network (WWARN) protocols. 0.2 to 0.5 mL of packed red blood cells (pRBC) from each sample was homogenized by mixing with 10 volumes of TRIzol (Invitrogen), frozen at -80oC.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using Trizol and ZYMO DirectZol96 extraction kit following protocol described in Kucharski, M., Tripathi, J., Nayak, S. et al. A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples. Malar J 19, 363 (2020).
Reverse transcription and cDNA synthesis was done following modifed Smart-Seq2 protocol using only oligo(d)T primers to enrich for mRNA transcripts (Kucharski, M., Tripathi, J., Nayak, S. et al. A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples. Malar J 19, 363 (2020)). Purified amplicon (cDNA) was used to generate sequencing libraries using Illumina Nextera XT kit as described in manufacturer’s protocol. Libraries were analyzed on the Bioanalyser High-Sensitivity DNA chips (Agilent, Cat. No.: 5067-4626), subsequently pooled (24 samples per lane) and sequenced on Illumina HiSeq4000 platform generating 150 bp paired end reads with 110 Gb data output generated per lane.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

P.falciparum parasite isolate
H0_sample12

Data processing

Standard Illumina pipeline was used for base calling and demultiplexing
Sequenced reads were trimmed for adapter, primer and low quality bases from 3'-ends using Trimgalore (version 0.4.4)
Trimmed reads then mapped to Plasmodium falciparum 3D7 genome (genedb version 3) using HiSAT2 (version 2.1.0) with parameters --min-intronlen 60 --max-intronlen 2500 --fr
Uniquely mapped and properly oridented reads were extracted using samtools (version: 1.8) and raw count generated by bedtools (v2.27.1)
Normalized count - Fragments Per Kilobase of transcript per Million mapped reads (FPKM) were calculated, except gene length in the formula is replaced with mapped length
Genome_build: Genome_build: Plasmodium falciparum 3D7 geneDB version 3
Supplementary_files_format_and_content: Tab-delimited text file includes Log2 FPKM values for each sample

Submission date

Mar 24, 2021

Last update date

Feb 07, 2022

Contact name

Zbynek Bozdech

E-mail(s)

ZBozdech@ntu.edu.sg

Organization name

Nanyang Technological University

Department

School of Biological Sciences