Transcriptome sequencing of the endangered land snail Karaftohelix adamsi from the Island Ulleung: De novo assembly, annotation, valuation of fitness genes and SSR markers

Park Jie Eun,Patnaik Bharat Bhusan,Sang Min Kyu,Song Dae Kwon,Jeong Jun Yang,Hong Chan Eui,Kim Yong Tae,Shin Hyeon Jun,Ziwei Liu,Patnaik Hongray Howrelia,Hwang Hee Ju,Park So Young,Kang Se Won,Ko Jung 한국유전학회 2024 Genes & Genomics Vol.46 No.7

Background The Bradybaenidae snail Karaftohelix adamsi is endemic to Korea, with the species tracked from Island Ulleung in North Gyeongsang Province of South Korea. K. adamsi has been classified under the Endangered Wildlife Class II species of Korea and poses a severe risk of extinction following habitat disturbances. With no available information at the DNA (genome) or mRNA (transcriptome) level for the species, conservation by utilizing informed molecular resources seems difficult. Objective In this study, we used the Illumina short-read sequencing and Trinity de novo assembly to draft the reference transcriptome of K. adamsi. Results After assembly, 13,753 unigenes were obtained of which 10,511 were annotated to public databases (a maximum of 10,165 unigenes found homologs in PANM DB). A total of 6,351, 3,535, 358, and 3,407 unigenes were ascribed to the functional categories under KOG, GO, KEGG, and IPS, respectively. The transcripts such as the HSP 70, aquaporin, TLR, and MAPK, among others, were screened as putative functional resources for adaptation. DNA transposons were found to be thickly populated in comparison to retrotransposons in the assembled unigenes. Further, 2,164 SSRs were screened with the promiscuous presence of dinucleotide repeats such as AC/GT and AG/CT. Conclusion The transcriptome-guided discovery of molecular resources in K. adamsi will not only serve as a basis for functional genomics studies but also provide sustainable tools to be utilized for the protection of the species in the wild. Moreover, the development of polymorphic SSRs is valuable for the identification of species from newer habitats and cross-species genotyping. Background The Bradybaenidae snail Karaftohelix adamsi is endemic to Korea, with the species tracked from Island Ulleung in North Gyeongsang Province of South Korea. K. adamsi has been classified under the Endangered Wildlife Class II species of Korea and poses a severe risk of extinction following habitat disturbances. With no available information at the DNA (genome) or mRNA (transcriptome) level for the species, conservation by utilizing informed molecular resources seems difficult. Objective In this study, we used the Illumina short-read sequencing and Trinity de novo assembly to draft the reference transcriptome of K. adamsi. Results After assembly, 13,753 unigenes were obtained of which 10,511 were annotated to public databases (a maximum of 10,165 unigenes found homologs in PANM DB). A total of 6,351, 3,535, 358, and 3,407 unigenes were ascribed to the functional categories under KOG, GO, KEGG, and IPS, respectively. The transcripts such as the HSP 70, aquaporin, TLR, and MAPK, among others, were screened as putative functional resources for adaptation. DNA transposons were found to be thickly populated in comparison to retrotransposons in the assembled unigenes. Further, 2,164 SSRs were screened with the promiscuous presence of dinucleotide repeats such as AC/GT and AG/CT. Conclusion The transcriptome-guided discovery of molecular resources in K. adamsi will not only serve as a basis for functional genomics studies but also provide sustainable tools to be utilized for the protection of the species in the wild. Moreover, the development of polymorphic SSRs is valuable for the identification of species from newer habitats and cross-species genotyping.

Comparison of library construction kits for mRNA sequencing in the Illumina platform

박용수,김송미,박동국,김동희,윤경욱,신원석,한규동 한국유전학회 2019 Genes & Genomics Vol.41 No.10

Background The emergence of next-generation sequencing (NGS) technologies has made a tremendous contribution to the deciphering and significance of transcriptome analysis in biological fields. Since the advent of NGS technology in 2007, Illumina, Inc. has provided one of the most widely used sequencing platforms for NGS analysis. Objective Although reagents and protocols provided by Illumina are adequately performed in transcriptome sequencing, recently, alternative reagents and protocols which are relatively cost effective are accessible. However, the kits derived from various manufacturers have advantages and disadvantages when researchers carry out the transcriptome library construction. Methods We compared them using a variety of protocols to produce Illumina-compatible libraries based on transcriptome. Three different mRNA sequencing kits were selected for this study: TruSeq ® RNA Sample Preparation V2 (Illumina, Inc., USA), Universal Plus mRNA-Seq (NuGEN, Ltd., UK), and NEBNext ® Ultra™ Directional RNA Library Prep Kit for Illumina ® (New England BioLabs, Ltd., USA). We compared them focusing on cost, experimental time, and data output. Results The quality and quantity of sequencing data obtained through the NGS technique were strongly influenced by the type of the sequencing library kits. It suggests that for transcriptome studies, researchers should select a suitable library construction kit according to the goal and resources of experiments. Conclusion The present work will help researchers to choose the right sequencing library construction kit for transcriptome analyses.

Transcriptome analysis and metabolic profiling of green and red kale (<i>Brassica oleracea</i> var. <i>acephala</i>) seedlings

Jeon, Jin,Kim, Jae Kwang,Kim, HyeRan,Kim, Yeon Jeong,Park, Yun Ji,Kim, Sun Ju,Kim, Changsoo,Park, Sang Un Elsevier 2018 Food chemistry Vol.241 No.-

<P><B>Abstract</B></P> <P>Kale (<I>Brassica oleracea</I> var. <I>acephala</I>) is a rich source of numerous health-benefiting compounds, including vitamins, glucosinolates, phenolic compounds, and carotenoids. However, the genetic resources for exploiting the phyto-nutritional traits of kales are limited. To acquire precise information on secondary metabolites in kales, we performed a comprehensive analysis of the transcriptome and metabolome of green and red kale seedlings. Kale transcriptome datasets revealed 37,149 annotated genes and several secondary metabolite biosynthetic genes. HPLC analysis revealed 14 glucosinolates, 20 anthocyanins, 3 phenylpropanoids, and 6 carotenoids in the kale seedlings that were examined. Red kale contained more glucosinolates, anthocyanins, and phenylpropanoids than green kale, whereas the carotenoid contents were much higher in green kale than in red kale. Ultimately, our data will be a valuable resource for future research on kale bio-engineering and will provide basic information to define gene-to-metabolite networks in kale.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The 49,471 transcripts were identified in kale transcriptome datasets. </LI> <LI> Sixty-eight secondary metabolite biosynthetic genes were identified in transcriptome. </LI> <LI> Forty-three secondary metabolites were detected in green and red kale seedlings. </LI> <LI> Some secondary metabolites were positively correlated with its biosynthetic genes. </LI> <LI> Most secondary metabolites were higher in red kale than in green kale. </LI> </UL> </P>

Transcriptome Profiling of Two Ornamental and Medicinal <i>Papaver</i> Herbs

Oh, Jaehyeon,Shin, Younhee,Ha, In Jin,Lee, Min Young,Lee, Seok-Geun,Kang, Byeong-Chul,Kyeong, Dongsoo,Kim, Dowan MDPI 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.10

<P>The <I>Papaver</I> spp. (<I>Papaver rhoeas</I> (Corn poppy) and <I>Papaver nudicaule</I> (Iceland poppy)) genera are ornamental and medicinal plants that are used for the isolation of alkaloid drugs. In this study, we generated 700 Mb of transcriptome sequences with the PacBio platform. They were assembled into 120,926 contigs, and 1185 (82.2%) of the benchmarking universal single-copy orthologs (BUSCO) core genes were completely present in our assembled transcriptome. Furthermore, using 128 Gb of Illumina sequences, the transcript expression was assessed at three stages of <I>Papaver</I> plant development (30, 60, and 90 days), from which we identified 137 differentially expressed transcripts. Furthermore, three co-occurrence heat maps are generated from 51 different plant genomes along with the <I>Papaver</I> transcriptome, i.e., secondary metabolite biosynthesis, isoquinoline alkaloid biosynthesis (BIA) pathway, and cytochrome. Sixty-nine transcripts in the BIA pathway along with 22 different alkaloids (quantified with LC-QTOF-MS/MS) were mapped into the BIA KEGG map (map00950). Finally, we identified 39 full-length cytochrome transcripts and compared them with other genomes. Collectively, this transcriptome data, along with the expression and quantitative metabolite profiles, provides an initial recording of secondary metabolites and their expression related to <I>Papaver</I> plant development. Moreover, these profiles could help to further detail the functional characterization of the various secondary metabolite biosynthesis and <I>Papaver</I> plant development associated problems.</P>

Full-length transcriptome combined with RNA sequence analysis of Fraxinus chinensis

Sun Xiaochun,Li Huirong 한국유전학회 2023 Genes & Genomics Vol.45 No.5

Background The dry root or stem bark of Fraxinus chinensis is a famous herb Qin Pi which is known for its anti-inflammatory, analgesic, anti-tumor, liver protective and diuretic pharmacological effects, the fundamental chemical components are coumarin, phenylethanol glycosides and flavonoids. However, it is difficult to clarify the secondary metabolite synthesis pathway and key genes involved in the pathway because of lack genome information of Fraxinus chinensis. Objective To generate a complete transcriptome of Fraxinus chinensis and to clarify the differentially expressed genes (DEGs) in leaves and stem barks. Methods In this study, full-length transcriptome analysis and RNA-Seq were combined to characterize Fraxinus chinensis transcriptome. Results A total of 69,145 transcripts were acquired and regarded as reference transcriptome, 67,441 transcripts (97.47%) were annotated to NCBI non-redundant protein (Nr), SwissProt, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and eukaryotic orthologous groups (KOG) databases. A total of 18,917 isoforms were annotated to KEGG database and classified to 138 biological pathways. In total, 10,822 simple sequence repeat (SSRs) and 11,319 resistance (R) gene were classified to 18 types, and 3947 transcription factors (TFs) were identified in full-length transcriptome analysis. Additionally, 15,095 DEGs were detected by RNA-seq in leaves and barks, including 4696 significantly up-regulated and 10,399 significantly down-regulated genes. And 254 transcripts were annotated into phenylpropane metabolism pathway containing 86 DEGs and ten of these enzyme genes were verified by qRT-PCR. Conclusion It laid the foundation for further exploration of the biosynthetic pathway of phenylpropanoids and related key enzyme genes.

Blood transcriptome resources of chinstrap (Pygoscelis antarcticus) and gentoo (Pygoscelis papua) penguins from the South Shetland Islands, Antarctica

김보미,정지혜,조은아,안도환,김정훈,이재성,박현 한국유전체학회 2019 Genomics & informatics Vol.17 No.1

The chinstrap (Pygoscelis antarcticus) and gentoo (P. papua) penguins are distributed throughout Antarctica and the sub-Antarctic islands. In this study, high-quality de novo assemblies of blood transcriptomes from these penguins were generated using the Illumina MiSeq platform. A total of 22.2 and 21.8 raw reads were obtained from chinstrap and gentoo penguins, respectively. These reads were assembled using the Oases assembly platform and resulted in 26,036 and 21,854 contigs with N50 values of 929 and 933 base pairs, respectively. Functional gene annotations through pathway analyses of the Gene Ontology, EuKaryotic Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were performed for each blood transcriptome, resulting in a similar compositional order between the two transcriptomes. Ortholog comparisons with previously published transcriptomes from the Adélie (P. adeliae) and emperor (Aptenodytes forsteri) penguins revealed that a high proportion of the four penguins’ transcriptomes had significant sequence homology. Because blood and tissues of penguins have been used to monitor pollution in Antarctica, immune parameters in blood could be important indicators for understanding the health status of penguins and other Antarctic animals. In the blood transcriptomes, KEGG analyses detected many essential genes involved in the major innate immunity pathways, which are key metabolic pathways for maintaining homeostasis against exogenous infections or toxins. Blood transcriptome studies such as this may be useful for checking the immune and health status of penguins without sacrifice.

Blood transcriptome resources of chinstrap (Pygoscelis antarcticus) and gentoo (Pygoscelis papua) penguins from the South Shetland Islands, Antarctica

Kim, Bo-Mi,Jeong, Jihye,Jo, Euna,Ahn, Do-Hwan,Kim, Jeong-Hoon,Rhee, Jae-Sung,Park, Hyun Korea Genome Organization 2019 Genomics & informatics Vol.17 No.1

The chinstrap (Pygoscelis antarcticus) and gentoo (P. papua) penguins are distributed throughout Antarctica and the sub-Antarctic islands. In this study, high-quality de novo assemblies of blood transcriptomes from these penguins were generated using the Illumina MiSeq platform. A total of 22.2 and 21.8 raw reads were obtained from chinstrap and gentoo penguins, respectively. These reads were assembled using the Oases assembly platform and resulted in 26,036 and 21,854 contigs with N50 values of 929 and 933 base pairs, respectively. Functional gene annotations through pathway analyses of the Gene Ontology, EuKaryotic Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were performed for each blood transcriptome, resulting in a similar compositional order between the two transcriptomes. Ortholog comparisons with previously published transcriptomes from the $Ad{\acute{e}}lie$ (P. adeliae) and emperor (Aptenodytes forsteri) penguins revealed that a high proportion of the four penguins' transcriptomes had significant sequence homology. Because blood and tissues of penguins have been used to monitor pollution in Antarctica, immune parameters in blood could be important indicators for understanding the health status of penguins and other Antarctic animals. In the blood transcriptomes, KEGG analyses detected many essential genes involved in the major innate immunity pathways, which are key metabolic pathways for maintaining homeostasis against exogenous infections or toxins. Blood transcriptome studies such as this may be useful for checking the immune and health status of penguins without sacrifice.

Genomic insights of Leclercia adecarboxylata strains linked to an outbreak in public hospitals in Mexico

Barrios-Villa Edwin,Pacheco-Flores Brenda,Lozano-Zaraín Patricia,Del Campo-Ortega Rodolfo,de Jesús Ascencio-Montiel Ivan,González-León Margot,Camorlinga-Ponce Margarita,Gaytán Cervantes Francisco Javi 한국유전학회 2023 Genes & Genomics Vol.45 No.5

Background The dry root or stem bark of Fraxinus chinensis is a famous herb Qin Pi which is known for its anti-inflammatory, analgesic, anti-tumor, liver protective and diuretic pharmacological effects, the fundamental chemical components are coumarin, phenylethanol glycosides and flavonoids. However, it is difficult to clarify the secondary metabolite synthesis pathway and key genes involved in the pathway because of lack genome information of Fraxinus chinensis. Objective To generate a complete transcriptome of Fraxinus chinensis and to clarify the differentially expressed genes (DEGs) in leaves and stem barks. Methods In this study, full-length transcriptome analysis and RNA-Seq were combined to characterize Fraxinus chinensis transcriptome. Results A total of 69,145 transcripts were acquired and regarded as reference transcriptome, 67,441 transcripts (97.47%) were annotated to NCBI non-redundant protein (Nr), SwissProt, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and eukaryotic orthologous groups (KOG) databases. A total of 18,917 isoforms were annotated to KEGG database and classified to 138 biological pathways. In total, 10,822 simple sequence repeat (SSRs) and 11,319 resistance (R) gene were classified to 18 types, and 3947 transcription factors (TFs) were identified in full-length transcriptome analysis. Additionally, 15,095 DEGs were detected by RNA-seq in leaves and barks, including 4696 significantly up-regulated and 10,399 significantly down-regulated genes. And 254 transcripts were annotated into phenylpropane metabolism pathway containing 86 DEGs and ten of these enzyme genes were verified by qRT-PCR. Conclusion It laid the foundation for further exploration of the biosynthetic pathway of phenylpropanoids and related key enzyme genes.

RNA sequencing of the nephron transcriptome: a technical note

( Jae Wook Lee ) 대한신장학회 2015 Kidney Research and Clinical Practice Vol.34 No.4

To understand the functions of the kidney, the transcriptome of each part of the nephron needs to be profiled using a highly sensitive and unbiased tool. RNA sequencing (RNA-seq) has revolutionized transcriptomic research, enabling researchers to define transcription activity and functions of genomic elements with unprecedented sensitivity and precision. Recently, RNA-seq for polyadenylated messenger RNAs [poly(A)0-mRNAs] and classical microdissection were successfully combined to investigate the transcriptome of glomeruli and 14 different renal tubule segments. A rat kidney is perfused with and incubated in collagenase solution, and the digested kidney was manually dissected under a stereomicroscope. Individual glomeruli and renal tubule segments are identified by their anatomical and morphological characteristics and collected in phosphate-buffered saline. Poly(A)0- tailed mRNAs are released from cell lysate, captured by oligo-dT primers, and made into complementary DNAs (cDNAs) using a highly sensitive reverse transcription method. These cDNAs are sheared by sonication and prepared into adapter-ligated cDNA libraries for Illumina sequencing. Nucleotide sequences reported from the sequencing reaction are mapped to the rat reference genome for gene expression analysis. These RNA-seq transcriptomic data were highly consistent with prior knowledge of gene expression along the nephron. The gene expression data obtained in this work are available as a public Web page (https://helixweb.nih.gov/ ESBL/Database/NephronRNAseq/) and can be used to explore the transcriptomic landscape of the nephron.

De novo assembly, gene annotation, and molecular marker development using Illumina paired-end transcriptome sequencing in the clam Saxidomus purpuratus

Hongjun Li,Min Liu,Sheng Ye,Feng Yang 한국유전학회 2017 Genes & Genomics Vol.39 No.6

The clam Saxidomus purpuratus is an important economic marine bivalve species in China. In this study, we performed a de novo transcriptome sequencing of S. purpuratus gill tissues to generate a transcriptome dataset by performing Illumina paired-end sequencing on the HiSeq 2500 platform. A total of 10,488,024 raw reads were produced. After removing the inferior sequences, adaptor sequences, and rRNAs, 68,080,636 high-quality reads were obtained, from which 5,115,494 assembled contigs were produced. These contigs were assembled into 120,479 transcripts and 66,388 unigenes with mean lengths of 674.96 and 562.70 bp, respectively. All unigene sequences were compared against the NCBI databases, and 26,781 unigenes were annotated. Both the gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that several functional genes were involved in the stress response, immune system, and oxidation reduction process. Meanwhile, 414 simple sequence repeats (SSRs) were identified in the S. purpuratus transcriptome. SSR validation via PCR confirmed that 26 primer pairs had expected products, and 13 primers proved to be polymorphism among 30 individuals. The S. purpuratus transcriptome advances the underlying molecular understanding of this economic shellfish and provides a basis for further exploring S. purpuratus genomics resources.