Computational protocol: Genome-wide mapping reveals single-origin chromosome replication in Leishmania, a eukaryotic microbe

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Protocol publication

[…] The DNA libraries were prepared using the Nextera® XT DNA Sample Preparation kit (Illumina), and subsequently sequenced using Illumina MiSeq paired-end 250-bp sequencing system (Illumina). The samples were multiplexed, with each of the early S, late S, G1 and G2 samples per species/strain sequenced in the same run to eliminate differences due to batch effects. The resulting data were analysed for quality control using FastQC [], then trimmed using fastq-mcf (ea-utils []) to exclude the adapter sequences. The reads were next aligned to the respective reference genomes (TriTrypDB version 6.0) using Bowtie2 (version 2.2.0 --very-sensitive-local -k1) []. The aligned reads were then compared using essentially the method described previously [], but simplified to facilitate inter-species comparisons: reads were binned in 2.5-kb sections along each chromosome, and the number of reads in each bin then used to calculate the ratios between early S versus G2/G1 and late S versus G2 samples, scaled for the total size of the read library (reads per 2.5 kb per million reads mapped). These data were then represented in a graphical form using ggplot2 and the R package (version 3.0.2 []). Shell scripts used to generate these data are available from []. [...] A strategy employed previously [] was used, and the assays were planned according to MIQE guidelines []. Primers were designed for several regions across L. major chromosomes 8, 20, 29 and 36, as well as L. mexicana chromosomes 8 and 20, using Primer Express version 3.0 (BioRad), and according to suggested guidelines [] for primers to be used in qPCR. Primer sizes ranged from 17–24 bp, with melting temperatures from 58–60 °C, resulting in amplicons of 55–113 bp with melting temperatures from 79–85 °C. Primer efficiency and specificity were assessed for all pairs of primers by the analysis of calibration curves and melting profiles, respectively, which resulted in efficiencies of approximately 100 %, all within a 10 % interval. For normalization, the L. major gene LmjF.36.1980 (equivalent to LmxM.36.1980 in L. mexicana) was chosen as the reference gene, since the MFAseq data suggested it is in a non-origin region that is not yet replicated in early S phase. For each pair of primers, triplicates of each sample (early S, late S and G2 phases) were run per plate (MicroAmp® Optical 96-well Reaction Plate, Life Technologies), which were sealed with MicroAmp® clear adhesive film (Life Technologies). SYBR Select Master Mix (Life Technologies) was used, together with 400 nM of primers (Eurofins MWG Operon, Ebersberg, Germany) and 0.01 ng of sample gDNA, to a total of 20 μl per reaction. All experiments were run in a 7500 Real Time PCR system (Applied Biosystems), using the following PCR cycling conditions: 50 °C for 2 min and 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, 59 °C for 15 s, and 72 °C for 1 min. Fluorescence intensity data were collected at the end of the extension step (72 °C for 1 min), after which a final dissociation step was included in order to confirm the specificity of the reaction. The resulting fluorescence intensity data were then analysed by relative quantification using the ΔΔCt method [] (7500 software version 2.3, Applied Biosystems), with the G2 phase sample being used as the calibrator. Graphs were generated using GraphPad Prism version 5.03. Primers (Table ) targeting regions of the following genes (gene ID as presented in TritrypDB []) in L. major (LmjF) and L. mexicana (LmxM) were used: LmjF.29.0810, LmjF.29.0930, LmjF.29.0030, LmjF.29.2060, LmjF.08.0090, LmjF.08.1000, LmjF.08.0260, LmjF.08.0360, LmjF.36.1900, LmjF.36.3790, LmjF.36.2830, LmjF.36.3000, LmjF.20.0705, LmjF.20.1210, LmjF.20.1530; LmxM.08_29.0810, LmxM.08_29.0930, LmxM.08_29.0030, LmxM.08_29.2060, LmxM.08.0090, LmxM.08.1000, LmxM.08.0260, LmxM.08.0360, LmxM.36.1900, LmxM.36.3790, LmxM.36.2830, LmxM.36.3000, LmxM.20.0705, LmxM.20.1210, and LmxM.20.1530. […]

Pipeline specifications

Software tools FastQC, ea-utils, Bowtie2, Ggplot2, Primer Express
Databases TriTrypDB
Applications Miscellaneous, qPCR
Organisms Trypanosoma brucei