Construction of RNA-Seq Libraries from Large and Microscopic Tissues for the Illumina Sequencing Platform

Author(s):  
Hagop S. Atamian ◽  
Isgouhi Kaloshian
2018 ◽  
Author(s):  
Shengcai Liu ◽  
Liyun Peng ◽  
Junfei Pan ◽  
Xiao Wang ◽  
Chunli Zhao ◽  
...  

Betalains are abundant in amaranth plants. Additionally, the betalain molecular structure and metabolic pathway differ from those of betanin in beet plants. To date, only a few studies have examined the regulatory roles of miRNAs in betalain biosynthesis in plants. Thus, we constructed small RNA libraries for the red and green sectors of amaranth leaves to identify miRNAs associated with betalain biosynthesis. We identified 198 known and 41 novel miRNAs. Moreover, 216 miRNAs were distributed in 44 miRNA families, including miR156, miR159, miR160, miR166, miR172, miR319, miR167, miR396, and miR398. An analysis of all unigene sequences in an amaranth transcriptome database resulted in the detection of 493 target genes for the 239 screened miRNAs. The targets included SPL2, ARF18, ARF6, and NAC. A quantitative real-time polymerase chain reaction validation of 20 miRNAs and nine target genes revealed expression-level differences between the red and green sectors of amaranth leaves. This study involved the application of an Illumina sequencing platform to identify miRNAs regulating betalain metabolism in amaranth plants. The data presented herein may provide insights into the molecular mechanisms underlying the regulation of betalain biosynthesis in amaranth and other plant species.


2018 ◽  
Author(s):  
Shengcai Liu ◽  
Liyun Peng ◽  
Junfei Pan ◽  
Xiao Wang ◽  
Chunli Zhao ◽  
...  

Betalains are abundant in amaranth plants. Additionally, the betalain molecular structure and metabolic pathway differ from those of betanin in beet plants. To date, only a few studies have examined the regulatory roles of miRNAs in betalain biosynthesis in plants. Thus, we constructed small RNA libraries for the red and green sectors of amaranth leaves to identify miRNAs associated with betalain biosynthesis. We identified 198 known and 41 novel miRNAs. Moreover, 216 miRNAs were distributed in 44 miRNA families, including miR156, miR159, miR160, miR166, miR172, miR319, miR167, miR396, and miR398. An analysis of all unigene sequences in an amaranth transcriptome database resulted in the detection of 493 target genes for the 239 screened miRNAs. The targets included SPL2, ARF18, ARF6, and NAC. A quantitative real-time polymerase chain reaction validation of 20 miRNAs and nine target genes revealed expression-level differences between the red and green sectors of amaranth leaves. This study involved the application of an Illumina sequencing platform to identify miRNAs regulating betalain metabolism in amaranth plants. The data presented herein may provide insights into the molecular mechanisms underlying the regulation of betalain biosynthesis in amaranth and other plant species.


2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Xueyi Dong ◽  
Luyi Tian ◽  
Quentin Gouil ◽  
Hasaru Kariyawasam ◽  
Shian Su ◽  
...  

Abstract Application of Oxford Nanopore Technologies’ long-read sequencing platform to transcriptomic analysis is increasing in popularity. However, such analysis can be challenging due to the high sequence error and small library sizes, which decreases quantification accuracy and reduces power for statistical testing. Here, we report the analysis of two nanopore RNA-seq datasets with the goal of obtaining gene- and isoform-level differential expression information. A dataset of synthetic, spliced, spike-in RNAs (‘sequins’) as well as a mouse neural stem cell dataset from samples with a null mutation of the epigenetic regulator Smchd1 was analysed using a mix of long-read specific tools for preprocessing together with established short-read RNA-seq methods for downstream analysis. We used limma-voom to perform differential gene expression analysis, and the novel FLAMES pipeline to perform isoform identification and quantification, followed by DRIMSeq and limma-diffSplice (with stageR) to perform differential transcript usage analysis. We compared results from the sequins dataset to the ground truth, and results of the mouse dataset to a previous short-read study on equivalent samples. Overall, our work shows that transcriptomic analysis of long-read nanopore data using long-read specific preprocessing methods together with short-read differential expression methods and software that are already in wide use can yield meaningful results.


2019 ◽  
Vol 76 (11) ◽  
pp. 801-807
Author(s):  
Yuan Yuan ◽  
Guosheng Zhao ◽  
Hongwei Ji ◽  
Bin Peng ◽  
Zhiguo Huang ◽  
...  

ObjectivesThe influence of commercial helium–oxygen saturation diving on divers’ gut microbiotas was assessed to provide dietary suggestion.MethodsFaecal samples of 47 divers working offshore were collected before (T1), during (T2) and after (T3) saturation diving. Their living and excursion depths were 55–134 metres underwater with a saturation duration of 12–31 days and PaO2 of 38–65 kPa. The faecal samples were examined through 16S ribosomal DNA amplicon sequencing based on the Illumina sequencing platform to analyse changes in the bacteria composition in the divers’ guts.ResultsAlthough the α and β diversity of the gut microbiota did not change significantly, we found that living in a hyperbaric environment of helium–oxygen saturation decreased the abundance of the genus Bifidobacterium, an obligate anaerobe, from 2.43%±3.83% at T1 to 0.79%±1.23% at T2 and 0.59%±0.79% at T3. Additionally, the abundance of some short-chain fatty acid (SCFA)-producing bacteria, such as Fusicatenibacter, Faecalibacterium, rectale group and Anaerostipes, showed a decreased trend in the order of before, during and after diving. On the contrary, the abundance of species, such as Lactococcus garvieae, Actinomyces odontolyticus, Peptoclostridium difficile, Butyricimonas virosa, Streptococcus mutans, Porphyromonas asaccharolytica and A. graevenitzii, showed an increasing trend, but most of them were pathogens.ConclusionsOccupational exposure to high pressure in a helium–oxygen saturation environment decreased the abundance of Bifidobacterium and some SCFA-producing bacteria, and increased the risk of pathogenic bacterial infection. Supplementation of the diver diet with probiotics or prebiotics during saturation diving might prevent these undesirable changes.


BioTechniques ◽  
2011 ◽  
Vol 50 (3) ◽  
pp. 177-181 ◽  
Author(s):  
Steven R. Head ◽  
H.Kiyomi Komori ◽  
G.Traver Hart ◽  
John Shimashita ◽  
Lana Schaffer ◽  
...  

2019 ◽  
Vol 40 (1) ◽  
pp. 76-81 ◽  
Author(s):  
Shaobin Fang ◽  
Renxie Wu ◽  
Xi Shi ◽  
Yin Zhang ◽  
Mhd Ikhwanuddin ◽  
...  

Abstract We conducted a whole genome survey in the portunid crab Charybdis feriatus (Linnaeus, 1758) using Illumina sequencing platform and developed a set of polymorphic microsatellite loci. A total of 117.7 Gb of clean reads were generated, with 74× coverage of the estimated genome size of 1.4 Gb. The GC content, heterozygosity rate, and repeat sequence rate of the genome were estimated to be 40%, 1.1%, and 51%, respectively. A total of 3,779,209 microsatellites were identified from the genome. Sixty microsatellite loci were evaluated in a wild population of 40 individuals. As a result, 14 polymorphic microsatellite loci (23.3%) were obtained. The number of alleles (3 to 15), polymorphism information content (0.365 to 0.884), observed heterozygosity (0.050 to 0.975), and expected heterozygosity (0.450 to 0.907) per locus averaged 6.8, 0.652, 0.691, and 0.707, respectively. We show that the genome of C. feriatus has a high heterozygosity and repeat sequence rates, and provide a novel insight into the genome profile of marine crabs. The genetic markers developed in this study are potentially useful for studies on population dynamics and conservation genetics of C. feriatus and other species of brachyuran crabs.


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