Since transcriptome analysis provides genome-wide sequence and gene expression information, transcript reconstruction using RNA-Seq sequence reads has become popular during recent years. For non-model organism, as distinct from the reference genome-based mapping, sequence reads are processed via transcriptome assembly approaches to produce large numbers of contigs corresponding to coding or non-coding, but expressed, part of genome. In spite of immense potential of RNA-Seq–based methods, particularly in recovering full-length transcripts and spliced isoforms from short-reads, the accurate results can be only obtained by the procedures to be taken in a step-by-step manner. In this chapter, we aim to provide an overview of the state-of-the-art methods including (i) quality check and pre-processing of raw reads, (ii) the pros and cons of transcriptome assemblers, (iii) generating non-redundant transcript data, (iv) current quality assessment tools for transcriptome assemblies, (v) approaches for transcript abundance and differential expression estimations and finally (vi) further mining of transcriptomic data for particular biological questions. Our intention is to provide an overview and practical guidance for choosing the appropriate approaches to best meet the needs of researchers in this area and also outline the strategies to improve on-going projects.

The NCBI SRA database is constantly expanding due to the large amount of genomic and transcriptomic data from various organisms generated by next-generation sequencing, and re-searchers worldwide regularly deposit new data into the database. This high-coverage genomic and transcriptomic information can be re-evaluated regardless of the original research subject. The database-deposited NGS data can offer valuable insights into the genomes of organelles, particularly for non-model organisms. Here, we developed an automated bioinformatics workflow called “OrgaMiner”, designed to unveil high-quality mitochondrial and chloroplast genomes by data mining the NCBI SRA database. OrgaMiner, a Python-based pipeline, automatically orchestrates various tools to extract, assemble, and annotate organelle genomes for non-model organisms without available organelle genome sequences but with data in the NCBI SRA. To test the usability and feasibility of the pipeline, “mollusca” was selected as a keyword, and 76 new mitochondrial genomes were de novo assembled and annotated automatically without writing one single code. The applicability of the pipeline can be expanded to identify organelles in diverse invertebrate, vertebrate, and plant species by simply specifying the taxonomic name. OrgaMiner provides an easy-to-use, end-to-end solution for biologists mainly working with taxonomy and population genetics.

Crocus istanbulensis (B.Mathew) Rukšāns is one of the most endangered Crocus species in the world and has an extremely limited distribution range in Istanbul. Our recent field work indicates that no more than one hundred individuals remain in the wild. In the present study, we used genome skimming to determine the complete chloroplast (cp) genome sequences of six C. istanbulensis individuals collected from the locus classicus. The cp genome of C. istanbulensis has 151,199 base pairs (bp), with a large single-copy (LSC) (81,197 bp), small single copy (SSC) (17,524 bp) and two inverted repeat (IR) regions of 26,236 bp each. The cp genome contains 132 genes, of which 86 are protein-coding (PCGs), 8 are rRNA and 38 are tRNA genes. Most of the repeats are found in intergenic spacers of Crocus species. Mononucleotide repeats were most abundant, accounting for over 80% of total repeats. The cp genome contained four palindrome repeats and one forward repeat. Comparative analyses among other Iridaceae species identified one inversion in the terminal positions of LSC region and three different gene (psbA, rps3 and rpl22) arrangements in C. istanbulensis that were not reported previously. To measure selective pressure in the exons of chloroplast coding sequences, we performed a sequence analysis of plastome-encoded genes. A total of seven genes (accD, rpoC2, psbK, rps12, ccsA, clpP and ycf2) were detected under positive selection in the cp genome. Alignment-free sequence comparison showed an extremely low sequence diversity across naturally occurring C. istanbulensis specimens. All six sequenced individuals shared the same cp haplotype. In summary, this study will aid further research on the molecular evolution and development of ex situ conservation strategies of C. istanbulensis.

As the most readily adopted molecular screening test, low-pass WGS of maternal plasma cell-free DNA for aneuploidy detection generates a vast amount of genomic data. This large-scale method also allows for high -throughput virome screening. NIPT sequencing data, yielding 6.57 terabases of data from 187.8 billion reads, from 12,951 pregnant Turkish women was used to investigate the prevalence and abundance of viral DNA in plasma. Among the 22 virus sequences identified in 12% of participants were human papillomavirus, herpes -virus, betaherpesvirus and anellovirus. We observed a unique pattern of circulating viral DNA with a high prevalence of papillomaviruses. The prevalence of herpesviruses/anellovirus was similar among Turkish, Eu-ropean and Dutch populations. Hepatitis B prevalence was remarkably low in Dutch, European and Turkish populations, but higher in China. WGS data revealed that herpesvirus/anelloviruses are naturally found in Eu-ropean populations. This represents the first comprehensive research on the plasma virome of pregnant Turkish women.

Bumblebees are crucial pollinators, providing essential ecosystem services and global food production. The success of pollination services relies on the interaction between sensory organs and the environment. The antenna functions as a versatile multi‐sensory organ, pivotal in mediating chemosensory/olfactory information, and governs adaptive responses to environmental changes. Despite an increasing number of RNA‐sequencing studies on insect antenna, comprehensive antennal transcriptome studies at the different life stages were not elucidated systematically. Here, we quantified the expression profile and dynamics of coding/microRNA genes of larval head and antennal tissues from early‐ and late‐stage pupa to the adult of Bombus terrestris as suitable model organism among pollinators. We further performed Pearson correlation analyses on the gene expression profiles of the antennal transcriptome from larval head tissue to adult stages, exploring both positive and negative expression trends. The positively correlated coding genes were primarily enriched in sensory perception of chemical stimuli, ion transport, transmembrane transport processes and olfactory receptor activity. Negatively correlated genes were mainly enriched in organic substance biosynthesis and regulatory mechanisms underlying larval body patterning and the formation of juvenile antennal structures. As post‐transcriptional regulators, miR‐1000‐5p, miR‐13b‐3p, miR‐263‐5p and miR‐252‐5p showed positive correlations, whereas miR‐315‐5p, miR‐92b‐3p, miR‐137‐3p, miR‐11‐3p and miR‐10‐3p exhibited negative correlations in antennal tissue. Notably, based on the inverse expression relationship, positively and negatively correlated microRNA (miRNA)–mRNA target pairs revealed that differentially expressed miRNAs predictively targeted genes involved in antennal development, shaping antennal structures and regulating antenna‐specific functions. Our data serve as a foundation for understanding stage‐specific antennal transcriptomes and large‐scale comparative analysis of transcriptomes in different insects.