2500 sequencer

Genomic DNA was extracted from whole flies for each of the six selected and six control populations in 2014, after ∼150 generations. We pooled 400 females per population and homogenized them in liquid nitrogen prior to pooled DNA extractions using the Qiagen Dneasy Blood and Tissue DNA extraction kit (Qiagen, Hilden, Germany) with modifications (Kapun et al. 2020 ). DNA of each sample was sheared in a Covaris instrument. Sample-wise library preparation following manufacturer’s instructions for paired-end sequencing were carried out using the Illumina TrueSeq Nano Library kit (Illumina, San Diego, CA). DNA pools were sequenced in two multiplexed batches of six samples (three selected and three control population per batch) each on an Illumina 2500 sequencer at the genomics technologies facility of the University of Lausanne, yielding paired-end sequences of 100 bp lengths.

The quality of RNA, and the extent of rRNA depletion, were assessed by running total and ribodepleted RNA on a 2100 Bioanalyzer system (Agilent) using the RNA pico kit. 10-30ng of ribodepleted high quality RNA was used for RNA-Seq library preparation with NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB) according to manufacturer’s instructions, except that a custom set of Y-shape adapters were used, harboring 6nt-long barcodes for sample multiplexing. PCR was performed with a universal primer pair (PE1.0 and PE2.0 primers from Illumina) for 14-15 cycles.
After library preparation, typically 4 libraries were multiplexed together. Equal molar amounts of each library were added to a single 0.5ml low-binding tube (Eppendorf) and the final volume was subjected to a purification/size-selection procedure using 1.4x AMPure XP beads (Beckam Coulter), to eliminate residual adaptor-dimer. Both individual and pooled libraries were assessed on a 2100 Bioanalyzer system using the DNA HS kit. Two biological replicates for each condition (three for the 6-8h unsorted) were sequenced on either a Illumina HiSeq 2000 or 2500 sequencer, using 100-bp paired-end reads. All sequencing was performed by the EMBL Genomics Core Facility. The number of mapped reads per sample (per replicate), is provided in Table S1.

RNA‐seq was carried out as previously described in (Ogrodnik et al,2017). Briefly, strand‐specific paired‐end libraries for RNA‐seq were generated from DNAse‐treated total RNA using Ribozero and ScriptSeq systems (Epicentre/Illumina) and run on an Illumina 2500 sequencer to obtain 100 base pair paired‐end reads with four libraries multiplexed per flow cell lane using 100 bp paired‐end reads. This resulted in an average of 250 million reads per lane, with an average of 40 million reads per sample. Each individual library received a unique Illumina barcode. Low‐quality reads were filtered out using Kraken (Davis et al,2013). The resulting filtered reads were mapped to the mouse genome version mm10 using STAR aligner (Dobin et al,2013) followed by estimates of raw gene counts using HTSeq (Anders et al,2015). Differential gene expression was analyzed using DESeq2 (Love et al,2014). Statistical significance was expressed as a P value adjusted for a false discovery rate of 0.01 using the Benjamini–Hochberg correction for multiple testing.

Tail snips from animals that showed the abnormal phenotype were assessed. Genomic DNA was extracted. This was fragmented to ~300bp and was processed for Illumina Next Gen DNA sequencing by standard library preparation protocols. The resulting library was DNA sequenced in one lane of an Illumina 2500 sequencer and yielded ~100 million paired end reads, each of 100 bases. The Fastq files from this were converted to a Fasta database. These were searched by BLAST for sequences homologous to the transgene (the Tie2 promoter, Cre cDNA, MT-1 polyA signal sequence, and Tie2 intron 1 enhancer). All of the resulting detected paired end sequence reads were extracted and further analyzed.

One hundred F2s from a backcross of each mutant to the N2;FR1gfp parent were transferred to individual plates and given time to lay eggs. Each F2 was used in single-worm DNA extractions to be genotyped later. Once the F3s reached the L4 stage, the segregation of their viral GFP expression phenotype was scored to determine if the F2 was wild type or a heterozygous or homozygous mutant. The F3 populations from 20 homozygous mutant F2s and 20 homozygous wild-type F2s were pooled, and DNA was extracted using the Gentra Puregene kit (Qiagen). The DNA was sheared using a Bioruptor (30 s on, 30 s off, for 15 min). To construct libraries for sequencing, 1 μg from each pool was used to generate a library using the PCR-free TruSeq DNA kit (Illumina). The samples were multiplexed to have six libraries in each lane for paired-end sequencing of 100-nt reads on an Illumina 2500 sequencer. Mutant and wild-type libraries for ucr2 were constructed using the NEXTflex PCR-free DNA library preparation kit for Illumina (Bioo Scientific), which gave lower genome coverage than the other three mutants. Mapping of the causal mutations in the four mutants by whole-genome sequencing and computational analysis was essentially as described previously (43 (link)).

Strand-specific paired-end libraries for RNA-Seq were generated from DNAse-treated total RNA using Ribozero and ScriptSeq systems (Epicentre/Illumina) and run on an Illumina 2500 sequencer to obtain 100 base paired-end reads. Low quality reads were filtered out by Kraken53 (link). The resulting filtered reads were mapped to the mouse genome version mm10 using Tophat54 (link). Mapped reads were counted with htseq-count55 (link) and read counts were normalized using deseq2 (ref. 56 (link)). In order to capture genes with the same expression pattern as the TAF and Oil Red O staining, said values were inserted into the normalized expression data set and then clustered with Biolayout express57 (link) using a 0.7 minimum Pearson correlation and a 95 correlation value. Clustering was conducted using MCL implementation of Markov Cluster Algorithm58 using an inflation coefficient of 2.2 and a preinflation coefficient of 3.0. The cluster of genes with included TAF and oil lipid data were extracted and analysed for GO over-representation using the PANTHER database59 (link).

Infection of N2 and mutant worms with OrV was done as described previously (9 (link), 14 (link)). Extraction of small RNAs and removal of the 5′-triphosphate groups from the secondary small RNAs by RNA 5′-polyphosphatase (RPP) were done as described previously (39 (link)). RPP-treated, purified small RNAs were used in the generation of small RNA libraries using the TruSeq small RNA sample preparation kit (Illumina). Samples were multiplexed and sequenced on an Illumina 2500 sequencer. The analysis of small RNAs was done as described previously (41 (link)). We removed the reads from each library that aligned with zero mismatches to the sense strand of structural small RNAs (25 (link)). The resultant nonstructural small RNA reads were used in the following analyses. The nonstructural small RNAs were aligned to C. elegans miRNAs obtained from WormBase (WS240) and used to normalize vsiRNA reads. Small RNA reads were aligned to the OrV genome (GenBank identifiers [IDs] HM030970.2 and HM030971.2), allowing zero mismatches essentially as described previously (41 (link)). For RPP-treated libraries, the sense vsiRNAs were normalized to the total sense vsiRNAs, whereas antisense reads were normalized to total vsiRNAs as described previously (14 (link)).