SARS-CoV-2 DNA library preparation using an adapted version of Illumina DNA prep protocol v.1.1 v1

Author(s):  
Emmanuel Kagning Tsinda ◽  
Masahiro Sakamoto ◽  
Michiko Okamoto ◽  
Clyde Dapat ◽  
Mariko Saito ◽  
...  

This protocol describes the steps to prepare DNA libraries from PCR amplicons using the Illumina DNA prep library kit. The current library preparation protocol was adapted from the original Illumina DNA Prep Reference Guide (document #1000000025416 v09) using low input DNA samples as the starting material. We omitted laboratory equipment such as the Eppendorf 96-well PCR plate, microseal adhesive seals. In addition, we replaced 96-well plate magnetic stand with a magnetic stand suitable for 1.5 ml tubes. The added value of this protocol is that PCR reactions happen in 0.2 ml PCR tubes, and it can be implemented without a separate purchase of 96-well PCR plates or a magnetic stand for PCR plates. Using individual 0.2 ml tubes increases the user flexibility when running few samples for library preparation. The other advantage of using our adapted protocol is the reduced library preparation cost when running few specimens. For instance, implementing the current protocol might be cheaper than the original Illumina protocol when using the Illumina® DNA Prep, (M) Tagmentation (24 Samples) catalog number 20018704. The protocol has proven effective for processing hundreds of DNA libraries from tiled virus amplicons such as Sapovirus and SARS-CoV-2 submitted to public repositories such as GISAID and GenBank.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15584-e15584
Author(s):  
Bosun Min ◽  
Ushati Das Chakravarty ◽  
Manqing Hong ◽  
Ekaterina Star ◽  
Hsiao-Yun Huang ◽  
...  

e15584 Background: Comprehensive tumor profiling using NGS is fundamentally transforming oncology research. However, converting archival tissue samples into libraries is often challenging due to the low quantity and quality of DNA. Here we present accurate detection of variants in the human exome using the novel chemistry of the xGen Prism DNA library preparation kit, optimized for low-input and degraded samples, with xGen Research Exome v2.0 hybrid-capture enrichment. Methods: The IDT Exome v2 panel was used to carry out targeted sequencing of Prism DNA libraries generated from archival FFPE samples. The unique library preparation is enabled by an engineered mutant ligase and proprietary adapters that prevent chimeras and suppress dimer-formation, thereby maximizing the conversion of input DNA to sequencing libraries. Results: We achieved high yields of library (300-400 ng) from input amounts as low as 25 ng for severely damaged FFPE samples (DIN 1-3), > 90% on-target rates and uniform depth of coverage ( > 96% bases covered at > 20X and > 98% bases covered at > 10X) for FFPE samples across a wide range in quality. We also observed minimal exon drop-outs in difficult-to-target genes for severely damaged FFPE material. To validate the variant calling performance of the Prism-Exome workflow, we used the Horizon OncoSpan FFPE reference control which contains 1-92% AF SNVs and Indels and achieved > 98% sensitivity across ~250 SNVs and Indels. Conclusions: This study demonstrates that the xGen Exome Research v2, when combined with xGen Prism DNA library preparation, provides researchers with a complete human exome FFPE-sequencing solution with robust performance across FFPE samples of varying quality.


Author(s):  
Djoko Soemarno ◽  
Surip Mawardi ◽  
Maspur Maspur ◽  
Henik Prayuginingsih

Ngada Residence is main producen region Arabica coffee in Nusa Tenggara Timur province. There are scattered on district of Bajawa and Golewa, that all of them effort by farmers and low quality, so farmers get low price and coffee development slowly than other coffee region in Indonesia. But, on the other hand, Arabica coffee from this region have potential special taste to be export quality coffee beans. One of way to solve to develop this quality is implementation coffee processing by Wet Process methode and support marketing system better by Model Kemitraan Bermediasi (Motramed). This research started from June until October 2007 at two centre district of Arabica coffee, there are district Bajawa are UPH Fa Masa on Beiwali village, UPH Wonga Wali on Susu village, UPH Papa Taki on Bomari village, UPH Suka Maju on Ubedolumolo village and Kecamatan Golewa are UPH Papa Wiu on Mangulewa village, UPH Meza Mogo on Rakateda II village and UPH Ate Riji on Were I village. This research want to know added value, cost efficiency, and profit on Arabica coffee processing used wet process methode on Unit Pengolahan Hasil (UPH) at Ngada Residence. Data was analysed by approximation added value, R-C Ratio analisys and t-One Sample Test. The result showed that Arabica coffee wet process could improved phisic and taste quality, lower of beans size, higher quality grade, smaller defect beans, moisture content lower, had special taste and very few taste defect. Those quality improvement improved price market to be higher, the added value about Rp4,390,- per kg and improved profit for farmers.Key words : Arabica coffee, wet process, quality, added value, efisiency, revenue.


2019 ◽  
Author(s):  
Andrea Sanchini ◽  
Christine Jandrasits ◽  
Julius Tembrockhaus ◽  
Thomas Andreas Kohl ◽  
Christian Utpatel ◽  
...  

AbstractIntroductionImproving the surveillance of tuberculosis (TB) is especially important for multidrug-resistant (MDR) and extensively drug-resistant (XDR)-TB. The large amount of publicly available whole-genome sequencing (WGS) data for TB gives us the chance to re-use data and to perform additional analysis at a large scale.AimWe assessed the usefulness of raw WGS data of global MDR/XDR-TB isolates available from public repositories to improve TB surveillance.MethodsWe extracted raw WGS data and the related metadata of Mycobacterium tuberculosis isolates available from the Sequence Read Archive. We compared this public dataset with WGS data and metadata of 131 MDR- and XDR-TB isolates from Germany in 2012-2013.ResultsWe aggregated a dataset that includes 1,081 MDR and 250 XDR isolates among which we identified 133 molecular clusters. In 16 clusters, the isolates were from at least two different countries. For example, cluster2 included 56 MDR/XDR isolates from Moldova, Georgia, and Germany. By comparing the WGS data from Germany and the public dataset, we found that 11 clusters contained at least one isolate from Germany and at least one isolate from another country. We could, therefore, connect TB cases despite missing epidemiological information.ConclusionWe demonstrated the added value of using WGS raw data from public repositories to contribute to TB surveillance. By comparing the German and the public dataset, we identified potential international transmission events. Thus, using this approach might support the interpretation of national surveillance results in an international context.


2021 ◽  
Vol 16 (2) ◽  
pp. 113-121
Author(s):  
Orléans Ngomo ◽  
Joseph Sieliechi ◽  
Etienne Dongo

Avocado is a versatile and valuable product. Its oil is comparable to olive oil in terms of nutritional quality. It can also be used in cosmetics, in particular, in soaps, shampoos and lotions. Despite all these important attributes that avocado has, it is highly perishable and coupled with the lack of farm-to-market roads, a lot of it is lost after harvest during the peak season. The introduction of methods that will transform avocado to products with a long shelf life and added value will go a long way to solving the problem of post-harvest losses and poverty. One of these methods is the production of avocado oil. In addition, in order to reduce the colour of the pigments without altering their quality, discoloration tests were carried out by adsorption on bleaching grounds (a montmorillonite, a kaolinite and activated carbon). The colour intensities of the oils before and after adsorption were determined using two complementary methods: a UV-Vis spectrophotometer and a Konica Minolta spectrophotometer CM-5. The UV-Vis spectrophotometer show that the activated carbon has a best fixing capacity of the pigments; According to the Colorimetric parameters (CIE-Lab) the coordinates L* a* b* that showed brightness (L*) of the avocado oil was half of the olive oil (41.13±0.02 vs 84.85±0.02). The activated carbon was better in fixing the red (a*) (4.99±0.01vs 15.73±0.01 before adsorption) and yellow (b*) (63.71±0.09 vs 70.07±0.09 before adsorption) pigments, while the other two adsorbents have very little influence on the red colour of avocado oil. RésuméL’avocat est un produit versatile et de grande valeur. Son huile est comparable à huile d’olive en terme de qualité nutritionnelle ; elle peut également être utilisée en cosmétique en occurrence dans les savons, champoings et lotions. En pleine saison, l’on fait face à une abondance des avocats dans les zones de production qui sont très souvent enclavées ; c’est ainsi que face aux difficultés de transport et les routes peu praticables pour l’importation, l’on se retrouve en train de perdre de très importantes quantités d’avocats après les récoltes. Or la production d’huile d’avocats permettrait de réduire les pertes post récolte, réduirait le chômage et permettrait de lutter contre la pauvreté après la vente des huiles extraites. De plus, dans le but de réduire les pigments colorés, des tests de décolorations effectués par adsorption sur des terres décolorantes (une montmorillonite en provenance de Maroua, une kaolinite en provenance de Douala et le charbon actif) sont effectués. Les couleurs des huiles mesurées avant et après adsorption à l’aide deux appareils complémentaires : Le spectrophotomètre UV-Vis et du Konica Minolta spectrophotomètre CM-5 montrent. Les analyses spectrophotométriques UV-Vis montrent que c’est le charbon activé qui présente la meilleure adsorption des pigments ; D’après les coordonnées L*a*b*, la clarté de l’huile d’avocat est pratiquement la moitié de celle de l’huile d’olive (41,13±0,02 contre 84,85±0,02), le charbon activé fixe mieux les pigments rouges (a*) (4,99±0,01 contre 15,73±0,01 avant adsorption) et jaunes (b*) (63,71±0,09 contre 70,07±0,09 après adsorption), tandis que les deux autres adsorbants influencent très peu sur la coloration des huiles.


2016 ◽  
Vol 11 (2) ◽  
pp. 144-152 ◽  
Author(s):  
Mariano Pernetti ◽  
Mauro D’Apuzzo Mauro D’Apuzzo ◽  
Francesco Galante

Vehicle speed is one of main parameters describing driver behavior and it is of paramount importance as it affects the travel safety level. Speed is, in turn, affected by several factors among which in-vehicle vibration may play a significant role. Most of speed reducing traffic calming countermeasures adopted nowadays rely on vertical vibration level perceived by drivers that is based on the dynamic interaction between the vehicle and the road roughness. On the other hand, this latter has to be carefully monitored and controlled as it is a key parameter in pavement managements systems since it influences riding comfort, pavement damage and Vehicle Operating Costs. There is therefore the need to analyse the trade-off between safety requirements and maintenance issues related to road roughness level. In this connection, experimental studies aimed at evaluating the potential of using road roughness in mitigating drivers’ speed in a controlled environment may provide added value in dealing with this issue. In this paper a new research methodology making use of a dynamic driver simulator operating at the TEST Laboratory in Naples is presented in order to investigate the relationship between the driver speed behavior on one hand, and the road roughness level, road alignment and environment, vehicle characteristics on the other. Following an initial calibration phase, preliminary results seem fairly promising since they comply with the published data derived from scientific literature.


2017 ◽  
Vol 8 (2) ◽  
pp. 323-330
Author(s):  
Rina Mirdayanti ◽  
Murni

The ability to use practical tools for students is a must in the basic Physics practical courses. In fact often happens is not adequate for laboratory tersediannya the implementation of practical, so that the process of teaching being constrained. To like the other problems like this needed a media device such as a software-based virtual lab simulations that can be used in the teaching of basic physics. This research aims to analyze the exact effort that can be done to overcome the lack of sedian laboratory equipment. This type of research is research use approach to the study of literature. The expected results of this literature is the emergence of an understanding of basic physics lab course in conducting with virtual laboratory as a means to address the issue of sedian in the real Labs. This virtual lab so that it can be the right solution for students and teachers in carrying out practical work.   Abstrak Kemampuan menggunakan alat-alat praktikum bagi siswa merupakan suatu keharusan dalam mata pelajaran Fisika Dasar. Pada kenyataannya yang sering terjadi adalah tidak tersediannya laboratorium yang memadai untuk pelaksanaan praktikum, sehingga proses praktikum menjadi terkendala. Untuk mensiasati permasalahan seperti ini dibutuhkan sebuah media seperti perangkat software laboratorium virtual berbasis simulasi yang bisa digunakan dalam praktikum  fisika dasar. Penelitian ini bertujuan untuk menganalisis upaya yang tepat yang dapat dilakukan untuk mengatasi ketidak-sedian  peralatan  laboratorium. Jenis penelitian yang gunakan adalah penelitian dengan pendekatan studi literatur. Hasil yang diharapkan dari literatur ini adalah munculnya pemahaman dalam melaksanakan praktikum fisika dasar dengan laboratorium virtual sebagai sarana untuk mengatasi persoalan ketidak-sedian laboratorium secara nyata. Sehingga laboratorium virtual ini dapat menjadi solusi yang tepat bagi siswa dan guru dalam melaksanakan praktikum. Kata kunci: Praktikum, Ilmu Sains, Laboratorium Virtual, Pemahaman Konsep


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