Nebulizer

Genomic DNA was sheared into fragments of 100–1,200 bp (Nebulizer, Illumina) and was made into a paired-end DNA sequencing (DNA-seq) library using the Genomic DNA Sample Preparation Kit (Illumina, San Diego, USA). Libraries were sequenced using the Genome Analyzer IIx (Illumina) at the High Throughput Genomics Unit at the University of Washington. Reads were aligned against the reference genome (L. donovani BPK282/Ocl4, cloned line from Nepal). Somy and copy number information for each chromosome were calculated independently using custom written perl script entitled “find_copy_number.pl” (see supplementary methods, Text S1). Single nucleotide polymorphism and small indels were called by inputting alignment files (bam) from all the four libraries into GATK software [28] (link). A thorough manual inspection revealed around 30% of variant calls were false positives or incorrectly genotyped. Calls were then validated manually and reassigned genotypes if necessary. All the validated variants that were consistent within each group (VL-SL and CL-SL) but different between them were then analyzed in detail to study the effects on protein coding genes using SNP EFF(v3.2) tool [29] (link). Further details concerning genomic library preparation and sequencing are presented in Supplementary Methods (Text S1).

Genomic DNA of the mutant was purified using DNeasy Blood & Tissue Kit (Qiagen) and the quality was checked by DNA electrophoresis and NanoDrop 1000 (Thermo Fisher Scientific) analysis. Genome sequencing was performed by Macrogen. The procedure described briefly: 2 μg genomic DNA was randomly sheared using a nebulizer (Illumina) and the ends were repaired using polynucleotide kinase and Klenow enzyme. The 5′-ends of the DNA fragments were phosphorylated and a single adenine base was added to the 3′-ends using Klenow exo⁺ (Illumina). Following ligation of a pair of Illumina adaptors to the repaired ends, the DNA was amplified in 10 cycles, using adaptor primers (Illumina), and fragments of around 150 bp were isolated using agarose gel electrophoresis. Sequencing libraries were quantified with a 2100 BioAnalyzer DNA 1000 chip (Agilent) as well as the Picogreen fluorescence assay (Invitrogen). Cluster generations were performed on an Illumina cluster station using 11 pmol of sequencing libraries. A total of 38 cycles of sequencing were carried out using the Illumina Genome Analyzer IIx system according to the manufacturer’s specifications. CLC Genomics Workbench was used for mapping the reads, SNP and DIP detection and identification of genomic rearrangement using the published genome sequence of L. Lactis MG136323 (link)24 (link) as the reference.

For the MBD-seq analysis, LCM-DNAs from GM, IM, and GT cells were fragmented to 100–500 bp by 44 psi of gas for 1 min through a nebulizer (Illumina, San Diego, CA) and then subjected to methylated DNA enrichment using a MethylMiner Methylated DNA Enrichment Kit (Invitrogen, Carlsbad, CA). Briefly, the methylated DNAs were precipitated from each 500 ng of fragmented LCM-DNAs via binding to the methyl-CpG binding domain of`the human MBD2 protein, which was coupled to magnetic Dynabeads. Then, the methylated fragments were eluted with High-Salt Elution Buffer (Invitrogen) and purified with a MinElute PCR Purification Kit (Qiagen). The methylated DNA fragments were ligated to a pair of adaptors for Illumina sequencing. The ligation products were size-fractioned on a 2% agarose gel to obtain 200–300 bp fragments and subjected to 18 cycles of PCR amplification. Each library was diluted to 8 pM, and 76 cycles of single-read sequencing was performed on an Illumina Genome Analyzer II.