scholarly journals Comparing library preparation methods for SARS-CoV-2 multiplex amplicon sequencing on the Illumina MiSeq platform

2020 ◽  
Author(s):  
Elizabeth M. Batty ◽  
Theerarat Kochakarn ◽  
Arporn Wangwiwatsin ◽  
Khajohn Joonlasak ◽  
Angkana T. Huang ◽  
...  
Keyword(s):  
Variant Calling ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Library Preparation ◽  
Preparation Methods ◽  
Base Calling ◽  
Sequencing Library ◽  
Miseq Platform ◽  
Downstream Analysis ◽  
Sequencing Library Preparation

AbstractGenomic surveillance has a key role in tracking the ongoing COVID-19 pandemic, but information on how different sequencing library preparation approaches affect the data produced are lacking. We compared three library preparation methods using both tagmentation (Nextera XT and Nextera Flex) and ligation-based (KAPA HyperPrep) approaches on both positive and negative samples to provide insights into any methodological differences between the methods, and validate their use in SARS-CoV-2 amplicon sequencing. We show that all three library preparation methods allow us to recover near-complete SARS-CoV-2 genomes with identical SNP calls. The Nextera Flex and KAPA library preparation methods gave better coverage than libraries prepared with Nextera XT, which required more reads to call the same number of genomic positions. The KAPA ligation-based approach shows the lowest levels of human contamination, but contaminating reads had no effect on the downstream analysis. We found some examples of library preparation-specific differences in minority variant calling. Overall our data shows that the choice of Illumina library preparation method has minimal effects on consensus base calling and downstream phylogenetic analysis, and suggests that all methods would be suitable for use if specific reagents are difficult to obtain.

scholarly journals A comparative study of ChIP-seq sequencing library preparation methods

BMC Genomics ◽  
2016 ◽  
Vol 17 (1) ◽  
Author(s):  
Arvind Y. M. Sundaram ◽  
Timothy Hughes ◽  
Shea Biondi ◽  
Nathalie Bolduc ◽  
Sarah K. Bowman ◽  
...  
Keyword(s):  
Comparative Study ◽  
Library Preparation ◽  
Preparation Methods ◽  
Sequencing Library ◽  
Sequencing Library Preparation

scholarly journals Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Milda Mickutė ◽  
Kotryna Kvederavičiūtė ◽  
Aleksandr Osipenko ◽  
Raminta Mineikaitė ◽  
Saulius Klimašauskas ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Regulatory Networks ◽  
Library Preparation ◽  
Rna Seq ◽  
Basic Principles ◽  
Cofactor Binding ◽  
Sequencing Library ◽  
Sequencing Library Preparation ◽  
Target Rna

Abstract Background Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3′ adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2′-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3′ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA. Results We showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3′ end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs. Conclusions Our findings provide a valuable resource for studies of the RNA-centred regulatory networks in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques.

Abstract 556: Highly complex DNA sequencing library preparation for cfDNA enrichment panels using a single-stranded approach

2021 ◽  
Author(s):  
Charles J. Vaske ◽  
Chris Troll ◽  
Camille Schwartz ◽  
Colin Naughton ◽  
Abdullah Mahmood Ali ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Library Preparation ◽  
Sequencing Library ◽  
Sequencing Library Preparation

scholarly journals A novel proof of concept for capturing the diversity of endophytic fungi preserved in herbarium specimens

2018 ◽  
Vol 374 (1763) ◽  
pp. 20170395 ◽  
Author(s):  
Barnabas H. Daru ◽  
Elizabeth A. Bowman ◽  
Donald H. Pfister ◽  
A. Elizabeth Arnold
Keyword(s):  
Species Interactions ◽  
Fungal Endophytes ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
Herbarium Specimens ◽  
Proof Of Concept ◽  
Ledum Palustre ◽  
Surface Contaminants ◽  
Symptomless Plant ◽  
Miseq Platform

Herbarium specimens represent important records of morphological and genetic diversity of plants that inform questions relevant to global change, including species distributions, phenology and functional traits. It is increasingly appreciated that plant microbiomes can influence these aspects of plant biology, but little is known regarding the historic distribution of microbes associated with plants collected in the pre-molecular age. If microbiomes can be observed reliably in herbarium specimens, researchers will gain a new lens with which to examine microbial ecology, evolution, species interactions. Here, we describe a method for accessing historical plant microbiomes from preserved herbarium specimens, providing a proof of concept using two plant taxa from the imperiled boreal biome ( Andromeda polifolia and Ledum palustre subsp . groenlandicum, Ericaceae). We focus on fungal endophytes, which occur within symptomless plant tissues such as leaves. Through a three-part approach (i.e. culturing, cloning and next-generation amplicon sequencing via the Illumina MiSeq platform, with extensive controls), we examined endophyte communities in dried, pressed leaves that had been processed as regular herbarium specimens and stored at room temperature in a herbarium for four years . We retrieved only one endophyte in culture, but cloning and especially the MiSeq analysis revealed a rich community of foliar endophytes. The phylogenetic distribution and diversity of endophyte assemblages, especially among the Ascomycota, resemble endophyte communities from fresh plants collected in the boreal biome. We could distinguish communities of endophytes in each plant species and differentiate likely endophytes from fungi that could be surface contaminants. Taxa found by cloning were observed in the larger MiSeq dataset, but species richness was greater when subsets of the same tissues were evaluated with the MiSeq approach. Our findings provide a proof of concept for capturing endophyte DNA from herbarium specimens, supporting the importance of herbarium records as roadmaps for understanding the dynamics of plant-associated microbial biodiversity in the Anthropocene. This article is part of the theme issue ‘Biological collections for understanding biodiversity in the Anthropocene’.

scholarly journals Method for improved Illumina sequencing library preparation using NuGEN Ovation RNA-Seq System

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