Four enzymatic DNA library preparation kits were compared for sequencing Shiga toxin-producing E. coli. All kits produced high-quality sequence data which performed equally well in the downstream analyses for surveillance and outbreak detection. Important differences were noted in the workflow user-friendliness and per sample cost.
Double-stranded flanking ends affect the folding kinetics and conformational equilibrium of G-quadruplexes forming sequences within the promoter of KIT oncogene
G-quadruplexes embedded within promoters play a crucial role in regulating gene expression.
KIT is a widely studied oncogene, whose promoter contains three G-quadruplex forming sequences, c-kit1, c-kit2 and c-kit*. For these sequences, available studies cover ensemble and single-molecule analyses, although for kit* the latter were limited to a study on a promoter domain comprising all of them.
Recently, c-kit2 has been reported to fold according to a multi-step process involving folding intermediates.
Here, by exploiting fluorescence resonance energy transfer, both in ensemble and at the single molecule level, we investigated the folding of expressly designed constructs in which, alike in the physiological context, either c-kit2 or c-kit* are flanked by double stranded DNA segments.
To assess whether the presence of flanking ends at the borders of the G-quadruplex affects the folding, we studied under the same protocols oligonucleotides corresponding to the minimal G-quadruplex forming sequences.
Data suggest that addition of flanking ends results in biasing both the final equilibrium state and the folding kinetics.
A previously unconsidered aspect is thereby unravelled, which ought to be taken into account to achieve a deeper insight of the complex relationships underlying the fine tuning of the gene-regulatory properties of these fascinating DNA structures.
Comparison of seven single cell whole genome amplification commercial kits using targeted sequencing
Advances in whole genome amplification (WGA) techniques enable understanding of the genomic sequence at a single-cell level. Demand for single-cell dedicated WGA kits (scWGA) has led to the development of several commercial kits. To this point, no robust comparison of all available kits was performed.
Here, we benchmark an economical essay, comparing all commercially available scWGA kits.
Our comparison is based on targeted sequencing of thousands of genomic loci, including highly mutable regions, from a large cohort of human single cells.
Using this approach we have demonstrated the superiority of Ampli1 in genome coverage and of RepliG in reduced error rate. In summary, we show that no single kit is optimal across all categories, highlighting the need for a dedicated kit selection in accordance with experimental requirements.
Target sequence capture in orchids: Developing a kit to sequence hundreds of single-copy loci
Premise: Understanding relationships among orchid species and populations is of critical importance for orchid conservation. Target sequence capture has become a standard method for extracting hundreds of orthologous loci for phylogenomics. Up-front cost and time associated with the design of bait sets make this method prohibitively expensive for many researchers.
Therefore, we designed a target capture kit to reliably sequence hundreds of orthologous loci across orchid lineages.
Methods: We designed an Orchidaceae target capture bait set for 963 single-copy genes identified in published orchid genome sequences. The bait set was tested on 28 orchid species, with representatives of the subfamilies Cypripedioideae, Orchidoideae, and Epidendroideae.
Results: Between 1,518,041 and 87,946,590 paired-end 150-base reads were generated for target-enriched genomic libraries. We assembled an average of 812 genes per library for Epidendroideae species and a mean of 501 genes for species in the subfamilies Orchidoideae and Cypripedioideae.
Furthermore, libraries had on average 107 of the 254 genes that are included in the Angiosperms353 bait set, allowing for direct comparison of studies using either bait set.
Discussion: The Orchidaceae963 kit will enable greater accessibility and utility of next-generation sequencing for orchid systematics, population genetics, and identification in the illegal orchid trade.
Variants in linkage status at D5S818 detected by multiple STR kits comparison and Sanger sequencing
Background: D5S818 discrepancies have been reported in forensic parental testing due to null alleles. However, more cases may be ignored since proportional null alleles were missed without detection of heredity discrepancy between parents and offspring.
Results: In this study, null allele 12 at D5S818 was detected by the PowerPlex® 21 System with a higher occurrence rate on the basis of review on 2824 samples from the 1282 routine cases in the Chinese Han population. Sequencing results revealed a novel variant of guanine (G) into adenine (A) in the 7th [AGAT] repeats in the core repeat region accompanied by rs1187948322 in the samples with null allele 12.
Conclusions: Forensic STR typing may benefit from this discovery: (1) primer design of CE profiling system could be improved for sensitive population and (2) polymorphic information could be enriched for the accuracy and precision of NGS genotyping system.
The Peak area of D5S818 was also analyzed through different commercial STR kits. It is suggested that more attention should be paid on observed homozygosity with reduced peak area, especially for the samples from Chinese Han population.
Keywords: D5S818; STR typing; linkage status; null alleles; variant.
Joining forces in Ochnaceae phylogenomics: a tale of two targeted sequencing probe kits
Premise: Both universal and family-specific targeted sequencing probe kits are becoming widely used for the reconstruction of phylogenetic relationships in angiosperms.
Within the pantropical Ochnaceae, we show that with careful data filtering, universal kits are equally as capable in resolving intergeneric relationships as custom probe kits.
Furthermore, we show the strength in combining data from both kits to mitigate bias and provide a more robust result to resolve evolutionary relationships.
Methods: We sampled 23 Ochnaceae genera and used targeted sequencing with two probe kits, the universal Angiosperms353 kit and a family-specific kit. We used maximum likelihood inference with a concatenated matrix of loci and multispecies-coalescence approaches to infer relationships in the family. We explored phylogenetic informativeness and the impact of missing data on resolution and tree support.
Results: For the Angiosperms353 data set, the concatenation approach provided results more congruent with those of the Ochnaceae-specific data set. Filtering missing data was most impactful on the Angiosperms353 data set, with a relaxed threshold being the optimum scenario. The Ochnaceae-specific data set resolved consistent topologies using both inference methods, and no major improvements were obtained after data filtering. Merging of data obtained with the two kits resulted in a well-supported phylogenetic tree.
Conclusions: The Angiosperms353 data set improved upon data filtering, and missing data played an important role in phylogenetic reconstruction. The Angiosperms353 data set resolved the phylogenetic backbone of Ochnaceae as equally well as the family-specific data set. All analyses indicated that both Sauvagesia L. and Campylospermum Tiegh. as currently circumscribed are polyphyletic and require revised delimitation.
Keywords: coalescence; custom; maximum likelihood; missing data; phylogenetic informativeness; probe kit; universal.
Forensic nanopore sequencing of STRs and SNPs using Verogen’s ForenSeq DNA Signature Prep Kit and MinION
The MinION nanopore sequencing device (Oxford Nanopore Technologies, Oxford, UK) is the smallest commercially available sequencer and can be used outside of conventional laboratories.
The use of the MinION for forensic applications, however, is hindered by the high error rate of nanopore sequencing.
One approach to solving this problem is to identify forensic genetic markers that can consistently be typed correctly based on nanopore sequencing. In this pilot study, we explored the use of nanopore sequencing for single nucleotide polymorphism (SNP) and short tandem repeat (STR) profiling using Verogen’s (San Diego, CA, USA) ForenSeq DNA Signature Prep Kit. Thirty single-contributor samples and DNA standard material 2800 M were genotyped using the Illumina (San Diego, CA, USA) MiSeq FGx and MinION (with R9.4.1 flow cells) devices.
EpiNext Bisulfite Sequencing Kit (Illumina) |
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| P-1056 | EpiGentek |
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pOET Sequencing Primers |
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| GWB-200100 | GenWay Biotech | 2 x 100 ul | Ask for price |
Sequencing Reaction Clean-Up Kit |
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| 34500 | Norgen Biotek Corp | 50 Preps | 77.4 EUR |
SequaGel Sequencing System 1L Kit - 1KIT |
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| NAT1136 | Scientific Laboratory Supplies | 1KIT | 187.65 EUR |
SEQUENCING UNIT 33X45 CM |
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| ESEQ1100-SYS | Consort | ea | 2060 EUR |
SequaGel Sequencing System 2.2L Kit - EACH |
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| NAT1138 | Scientific Laboratory Supplies | EACH | 318.6 EUR |
Sequencing Grade Chymotrypsin |
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| RE013 | SAB | 100ug | 309 EUR |
SEQUENCING SYSTEM 20X50 CM |
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| ESEQ1200-SYS | Consort | ea | 2060 EUR |
Trypin for Mass & Sequencing |
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| T9600-025 | GenDepot | 25ug | 76 EUR |
Trypin for Mass & Sequencing |
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| T9600-100 | GenDepot | 100ug | 133 EUR |
Trypin for Mass & Sequencing |
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| T9600-112 | GenDepot | 12x100ug | 1230 EUR |
With an optimized cutoff for allelic imbalance, all 94 identity-informative SNP loci could be genotyped reliably using the MinION device, with an overall accuracy of 99.958% (1 error among 2926 genotypes).
STR typing was notably error prone, and its accuracy was locus dependent.
We developed a custom-made bioinformatics workflow, and finally selected 13 autosomal STRs, 14 Y-STRs, and 4 X-STRs showing high consistency between nanopore and Illumina sequencing among the tested samples. These SNP and STR loci could be candidates for panel design for forensic analysis based on nanopore sequencing.