Genome analyzer iix gaiix

Total RNAs from five BCs and five NBEs were subjected to deep sequencing. The experimental process included the following steps: small RNA isolation, cDNA library preparation, and sequencing. Total RNA of each sample was size-fractionated on a 15% PAGE gel, and an 18–30 nucleotide fraction was collected. The 5′-RNA adapter was ligated to the RNA with T4 RNA ligase. Ligated RNA was size-fractionated, and the 36–50 nucleotide fraction was excised. The 3′-RNA adapter was subsequently ligated to precipitated RNA using T4 RNA ligase. Ligated RNA was size-fractionated, and the 62–75 nucleotide fraction (small RNA+adaptors) was excised. Small RNAs ligated with adaptors were subjected to RT-PCR to produce sequencing libraries. PCR products were purified and cDNA libraries, 106–118 bp in length, were collected. Then, cDNA libraries were sequenced by Genome Analyzer IIx (GAIIx) (Illumina Inc., CA, USA) at Beijing Genomics Institute at Shenzhen.

Mosquito DNA was extracted from single mosquitoes using the QIAamp DNA Mini Kit (Qiagen Inc. Valencia, CA) according to the manufacturer’s instructions. The extracted DNA was further cleaned and concentrated using DNA Clean & Concentrator (Zymo Research, Irvine, CA). DNA samples were quantified with Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies, Carlsbad, CA) and checked for quality by 1.0% agarose gel electrophoresis. All mosquitoes were genotyped for species identification by An. sinensis allele specific PCR (AS-PCR) following a previously published protocol (Joshi et al. 2010 (link)). High-quality DNA with equal amounts at the same concentration from each of the 20 resistant mosquitoes were pooled and named YLR (resistant). Similarly, equal amounts of high-quality DNA at the same concentration from each of the 20 susceptible mosquitoes were pooled and named YLS (susceptible). The two DNA pools were used to build paired-end libraries for whole genome DNA sequencing and sequenced on the Illumina Genome Analyzer IIx (GAIIx) at the Broad Institute of MIT and Harvard by running two lanes of PE100 sequencing (100-bp paired-end reads) per pool. Bases were called using Illumina software and data outputted as fastq files.

For the RNA-seq analysis, after integrity evaluation, 300 ng of RNA were used for library preparation with the TruSeq Stranded Total RNA LT Sample Prep kit with RiboZero (Illumina). The sequencing reaction proceeded as described before in the whole-exome sequencing: a 55-bp paired-end sequencing using the TruSeq SBS v5 Kit, in the Genome Analyzer IIx (GAIIx) Illumina platform. The software STAR was used to map the reads (GRCh38 -Gencode), count and annotate the transcripts^{50 (link)}. Normalization and differential expression analysis were performed with edgeR package in R^{51 (link)}. False discovery rate (FDR) was used to adjust p values. Genes with adjusted p value < 0.05 were considered differentially expressed. Volcanos plots and heatmaps were also generated in R.

For the sequencing of the A. faecalis MOR02 genome, sequencing on the Genome Analyzer IIx (GAIIx) Illumina platform was performed by the UUSMD (Unidad Universitaria de Secuenciación Masiva de DNA, Instituto de Biotecnología, UNAM). The 19,250,362 paired-end reads were assembled de novo using the SPAdes program (version 3.1.1) and 23 contigs were generated with a 315-fold median coverage depth.

We used Illumina’s Genome Analyzer IIx (GAIIx) for mRNA-sequencing by loading one sample per lane. For mRNA-sequencing, the library was diluted to 10 nM in EB buffer and then denatured using the Illumina protocol. The denatured libraries were diluted to 12 pM, followed by cluster generation on a single-end Genome Analyzer IIx (GAIIx) flow cell (v4) using an Illumina cBOT, according to the manufacturer's instructions. The Illumina GAIIx flow cell was run for 75 cycles using a single-read recipe (v4 sequencing kits) according to the manufacturer’s instructions.

For the HIV and HCV plasmids datasets processed in-house for training and testing respectively, the Illumina Genome Analyzer IIx (GAIIx) was used for the paired-end sequencing. Basecalling was done using Bustard (CASAVA 1.7.0), the Illumina default basecaller, producing after de-multiplexing two FASTQ [20 (link)] files per plasmid: FASTQ/1 (reads 1 of paired-end reads) and FASTQ/2 (reads 2 of paired-end reads, sequenced after paired-end turn), containing the nucleotide sequences with a quality score assigned to each nucleotide. Training of the classifier model was then done on an in silico plasmid mixture dataset which we created with known true variant percentages.

Coral branches in CHAOS buffer were incubated at 56 °C for 48 hours. After that, coral skeletons were removed and preserved in the event that future species confirmation using skeletal characters might be needed. After ethanol precipitation, pellets were dissolved in ALT buffer from a QIAGEN DNeasy blood and tissue kit (cat. 69504) and then they were digested with proteinase K for 48 hours at 56 °C. Samples were further purified according to QIAGEN kit instructions, including RNaseA treatment. At the end, DNAs were eluted from spin columns using AE buffer and kept at 4 °C. Library preparation for re-sequencing of DNA from each individual genome using Illumina platforms (500 bp insert size) was done according to the manufacturer’s instructions. Libraries were sequenced using an Illumina Genome Analyzer IIx (GAIIx) (75 bp paired-end reads) and Hiseq 2000 (100 bp paired-end reads). Raw sequence data were submitted to the DNA Databank of Japan (DDBJ) Sequence Read Archive (DRA) under accession number DRA003938 (BioProject PRJDB4188).