Solexa

Dermatophagoides microceras was purchased from Thermo Scientific™ ImmunoCAP™ Mite Allergens, and we consigned the samples to Welgene Biotech Co., LTD (Taiwan) for genomic DNA isolation and NGS sequencing by Illumina Solexa™ platform. The paired-end cleaned reads were assembled by ABySS (version 2.1) with 31 k-mer sizes on the Ubuntu 16.04 server. The sequences of allergen proteins of Dermatophagoides farina and Dermatophagoides pteronyssinus were obtained from the WHO/IUIS Allergen Nomenclature Sub-Committee website (http://www.allergen.org/ (accessed on 5 March 2022)), and they were used for homologous proteins blast by BLASTP 2.8.0+ algorithm. The contigs with similar sequences to the allergen proteins were further compared by GeneWise to identify their cDNA sequences of the putative allergen proteins of Der m.

The first set of 45 genomes was sequenced using a shotgun approach with the Illumina Solexa technology (paired-end 2 × 100 bp in HiSeq2000). The last 11 genomes were sequenced using a shotgun approach in a HiSeq3000 system (paired-end 2 × 150 bp) (Supplementary Table S2).
Read sequences were submitted to the European Nucleotide Archive (ENA) under BioProject accession number PRJEB42834, except for strain H222, for which reads had previously been deposited under accession number PRJEB28424 [14 (link)].
We initially intended to use the genome of strain E150 sequenced in 2004 by Génolevures as a reference (Dujon et al., 2004) [9 (link)]. However, by comparison with other Y. lipolytica available genomes, it appears that a quite large region was missing at the 3′ extremity of chromosome Yali0D. Thus, the Sanger reads produced by the Génoscope in 2004 were manually re-assembled in that part of the genome, and a 28,097 bp extension was added, which contains 12 protein-coding genes. The revised version of the genome sequence and annotation is provided at https://doi.org/10.57745/UZFAEN (accessed on 15 December 2022). In total, the E150 genome contains 6509 protein-coding genes, not including the 30 alternative splicing isoforms nor the 63 transposable elements with a coding sequence.

F81 cells cultured in 6-well plates (Costar) were infected with MEV at an input multiplicity (MOI) of 1 pfu/cell. Uninfected cells were maintained as a control. Twenty-four h later, the triplicate cultures were pooled, total RNA was extracted by Trizol reagent (Invitrogen) and small RNAs with length of 18–30 nt were separated by PAGE. Ten μg samples of the isolated RNAs were submitted to Solexa (Illumina) for sequencing as cDNA libraries. Duplicate sequences were eliminated from the initial data set. The resulting sets of unique reads were mapped onto the feline genome [26 (link),27 (link)] using the program Short Oligonucleotide Analysis Package (SOAP) [28 (link)]. Perfectly matched reads were also mapped onto the miRNAs of six reference species (Homo sapiens, Canis familiaris, Mus musculus, Rattus norvegicus, Bos taurus and Sus scrofa) listed in the Sanger miRBase (Release 18) using the Patscan tool [29 (link)] to identify homologs of known miRNAs.

To test the effects of the parental environment on seed transcripts, we constructed Illumina Solexa sequencing libraries of 24 self-fertilized dry, ungerminated seeds derived from the same lot of seeds used for the phenotyping. The seeds experienced 1 year of after-ripening before they were used for this experiment. For each genotype and parental environment, we had three biological replicates that consist of a single seed per replicate. For each library, mRNA was isolated from a single seed using Dynabeads mRNA DIRECT Kit from Invitrogen (Product # 610.2, Grand Island, NY) and fragmented using Ambion mRNA Fragmentation Kit (Product #AM8740, Grand Island, NY), followed by cDNA synthesis using random hexamer primers. Double stranded cDNA fragments were blunt-end repaired using Epicentre End Repair (Product #ER81050, Madison, WI) and added a single A to the blunt end using Klenow Fragment 3′-5′ exo-nuclease (NEB Product #M0212L, Ipswich, MA). Ilumina adaptors were ligated to the cDNA fragments using the Epicentre Fast-Link DNA Ligation Kit (Product #LK6201H, Madison, WI). Fragments between 200-400 bp were size selected by agarose gel and samples were indexed and enriched. The libraries were indexed using 12 indices and sequenced on two lanes using the Illumina GAIIx, which generated 76 bp single-end reads.

Chinese amphioxus, B. belcheri, were collected and reared in Beihai, Guangxi, China. The mRNAs from male adult amphioxus were extracted for Roche 454 sequencing (Lib-1). Two cDNA libraries were constructed from total RNAs of samples from different developmental stages, including fertilized egg, 2-cell, 16-cell, 64-cell, 128–256-cell, gastrula, neurula, hatch, 12 hours, 24 hours, 48 hours, male adult and female adult stages and were sequenced with Illumina Solexa paired-end sequencing (Lib-2 with normalization and Lib-3 without normalization). The RNA libraries from each stage were also sequenced through DGE-Seq by BGI as described in the Illumina DGE-Seq protocol. All sequencing data were submitted to the National Center for Biotechnology Information (NCBI) (BioProject: PRJNA310680).