A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers., azide free, Blogging, elisa, Goat, Hamster, Mammal, Mink, Mouse, Multiplexed shotgun genotyping for rapid and efficient genetic mapping., NGS QC Toolkit: a toolkit for quality control of next generation sequencing data., Opossum, Orangutan, pcr, Pig, Pigeon, Plant, Rat, Yeast

Sanger sequencing is no longer always necessary based on a single-center validation of 1109 NGS variants in 825 clinical exomes

Despite the improved accuracy of next-generation sequencing (NGS), it is extensively accepted that variants should be validated utilizing Sanger sequencing earlier than reporting. Validation of all NGS variants significantly will increase the turnaround time and prices of clinical analysis. We comprehensively assessed this want in 1109 variants from 825 clinical exomes, the biggest pattern set up to now assessed utilizing Illumina chemistry reported. With a concordance of 100%, we conclude that Sanger sequencing will be very helpful as an inner high quality management, however not a lot as a verification technique for high-quality single-nucleotide and small insertion/deletions variants.

Laboratories would possibly validate and set up their very own thresholds earlier than discontinuing Sanger affirmation research. We additionally develop and validate 23 copy quantity variations detected by exome sequencing in 20 samples, observing a concordance of 95.65%. The part of aneuploid embryos was considerably greater in superior age group than in youthful age group (68.9 vs 39.9%, P < 0.001). But there have been no statistically important variations in being pregnant price (43.5 vs 64.7%), clinical being pregnant price (39.1 vs 48.0%), implantation price (39.1 vs 51.0%), and miscarriage price (4.Three vs 7.8%) per embryo switch (ET) between the 2 teams.

Results recommend that the embryo-related issue performs a essential function in RIF. Maternal age doesn’t affect the implantation potential of euploid blastocysts. The NGS-based PGT-A involving trophectoderm biopsy is helpful for RIF sufferers of superior age by bettering their clinical outcomes. In conclusion, the NGS-based PGT-A involving trophectoderm biopsy could symbolize a helpful complement to the present RIF administration. Nonetheless, these findings ought to be additional validated in a well-designed randomized managed trial.

[NGS sequencing in barley breeding and genetic studies]

Barley (Hordeum vulgare L.) is the one of crucial cereal species used as meals and feed crops, in addition to for malting and alcohol manufacturing. At the top of the final century, conventional breeding methods have been complemented by the use of DNA markers. Molecular markers have additionally been used extensively for molecular genetic mapping and QTL evaluation. In 2012, the barley genome sequencing was accomplished, which offered a broad vary of new alternatives – from a extra environment friendly seek for candidate genes controlling economically vital traits to genomic choice.

The evaluate summarizes the outcomes of the research carried out after barley genome sequencing, which found new areas of barley genetics and breeding with excessive throughput screening and genotyping strategies. During this era, intensive research aimed toward identification of barley genomic loci related to economically vital traits have been carried out; on-line databases and instruments for working with barley genomic information and their deposition have appeared and are being replenished. In latest years, GWAS evaluation has been used for large-scale phenotypegenotype affiliation research

which has been extensively used in barley since 2010 because of the developed SNP-arrays, in addition to genotyping strategies based on direct NGS sequencing of chosen fractions of the genome. To date, greater than 80 papers have been revealed that describe the outcomes of the GWAS evaluation in barley. SNP identification related to economically vital traits and their transformation into CAPS or KASP markers handy for screening choice materials considerably expands the chances of marker-assisted choice of barley. In addition, the at present accessible info on potential goal genes and the standard of the entire barley genome sequence supplies a good base for making use of genome enhancing applied sciences to create materials for the creation of varieties with desired properties.

Sanger sequencing is no longer always necessary based on a single-center validation of 1109 NGS variants in 825 clinical exomes

Authentication, characterization and contamination detection of cell traces, xenografts and organoids by barcode deep NGS sequencing

Misidentification and contamination of biobank samples (e.g. cell traces) have plagued biomedical analysis. Short tandem repeat (STR) and single-nucleotide polymorphism assays are extensively used to authenticate biosamples and detect contamination, however with inadequate sensitivity at 5-10% and 3-5%, respectively. Here, we describe a deep NGS-based technique with considerably greater sensitivity (≤1%). It can be utilized to authenticate human and mouse cell traces, xenografts and organoids. We talk about accessible proof utilizing these molecular strategies for FUO evaluations, together with potential cost-benefits and future instructions.

It can even reliably determine and quantify contamination of human cell line samples, contaminated with solely small quantity of different cell samples; detect and quantify species-specific parts in human-mouse combined samples (e.g. xenografts) with 0.1% sensitivity; detect mycoplasma contamination; and infer inhabitants construction and gender of human samples. By adopting DNA barcoding know-how, we’re capable of profile 100-200 samples in a single run at per-sample value comparable to traditional STR assays, offering a actually high-throughput and low-cost assay for constructing and sustaining high-quality biobanks.

Even nicely into the 21st century, infectious ailments nonetheless account for many causes of fever of unknown origin (FUO). Advances in molecular applied sciences, together with broad-range PCR of the 16S rRNA gene adopted by Sanger sequencing, multiplex PCR assays, and extra lately, next-generation sequencing (NGS) purposes, have transitioned from analysis strategies to extra commonplace in some clinical microbiology laboratories. They have the potential to supplant conventional microbial identification strategies and antimicrobial susceptibility testing. Despite the remaining challenges with these applied sciences, publications in the previous decade justify pleasure in regards to the potential to remodel FUO investigations.


Highly sensitive ELISA for UV-induced DNA damage

The High Sensitivity CPD and the High Sensitivity (6-4) photoproducts ELISA from Cosmo Bio

Most of the UV-induced DNA damage are cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs), which are formed between neighboring pyrimidine nucleotides on the same DNA strand. These helix-distorting DNA lesions are only repaired in humans by a nucleotide excision repair system.The main types of UV-induced DNA damage are cyclobutane pyrimidine dimers (CPDs), (6-4) photoproducts (6-4PPs) and dewar photoproducts (DewarPPs), which are formed between neighboring pyrimidine nucleotides on the same DNA strand become.

Cyclobutane pyrimidine dimers (CPDs), (6-4) photoproducts (6-4PPs) and Dewar photoproducts (DewarPPs) are UV-induced DNA damage that are formed between neighboring pyrimidine nucleotides on the same DNA strand.

The High Sensitivity CPD / Cyclobutane Pyrimidine Dimer ELISA kit ( CSR-NM-MA-K003 ) from Cosmo Bio is the only commercially available ELISA using the anti-CPD clone TDM-2 and was developed for the highly sensitive detection of CPDs in cultured cells or DNA purified from the skin epidermis.

The High Sensitivity (6-4) photoproducts (6-4PPs) ELISA Kit ( CSR-NM-MA-K002 ) is the only commercially available ELISA that uses the anti-6-4PPs clone 64M-2 and for the highly sensitive one Detection of 6-4PPs from cultured cells from the skin epidermis was optimized.

This ELISA recognizes CPDs or 6-4PPs from dipyrimidines in all DNA sequence contexts (eg TT, TC, CT and CC). Therefore, the availability and convenience of this ELISA kit will help to understand the molecular mechanisms of cellular responses to UV radiation and DNA damage with applications in many areas of research such as cancer research, photobiology, dermatology, ophthalmology, immunology and cosmetic science.

To better investigate molecular events in the environment of UV-induced DNA damage and repair, Mori et al. previously developed and characterized monoclonal antibodies (mAb) specifically for CPDs and mAb specifically for 6-4PPs (1), while Matsunaga et al. mAb developed and characterized specifically for Dewar photo products (DewarPPs) (2).

Three of these antibodies (CPDs: clone TDM-2 [ CAC-NM-DND-001 ]; 6-4PPs: clone 64M-2 [ CAC-NM-DND-002 ]; DewarPPs: clone DEM-1 [ CAC-NM-DND -003 ]) continue to be widely cited in the literature, often for use in ELISA by a recommended method. These antibodies enable the quantification of photoproducts in purified DNA, those from cultured cells or from the skin epidermis using an enzyme-linked immunoadsorption test (ELISA) and the visualization and measurement of photoproducts in the DNA from cultivated cells or the skin by means of indirect immunofluorescence (IIF).


1) Mori, T., et al., Photochem. Photobiol. 54, 225-232 (1991).

2) Matsunaga, T., et al., Photochem. Photobiol. 54, 403-410 (1991).


Cell lines in the life sciences

Cell lines are an integral a part of the life sciences

Cell lines are discovering growing purposes in biology and the life sciences. They have a everlasting place in analysis and improvement. They are important in the manufacture of molecular merchandise akin to monoclonal antibodies, vaccines, enzymes and lots of others. In addition to those established purposes, they’re additionally used in new areas. One of those areas is “Cellular Agriculture”, which offers with the manufacturing of agricultural merchandise utilizing cell cultures.

Establishment of a cell line

The cultivation of cells takes place in a nutrient medium outdoors the organism. A distinction is made between immortalized lines and first cultures. The main tradition describes a non-immortalized cell tradition, which was obtained instantly from the tissue. In order to efficiently convey cells into tradition, the alternative of the tradition medium is of explicit significance. Among different issues, the pH worth and the buffer system have to be chosen appropriately. The cells should even be provided with the correct quantity of amino acids, nutritional vitamins and salts. Furthermore, the temperature and the oxygen and carbon dioxide focus have to be set optimally.

Frequently used cell lines

A complete of over 5000 cell lines are listed in the American Type Culture Collection (ATCC) and the European Collection of Cell Cultures (ECACC). There you possibly can request vital info and areas of utility to plan your individual analysis. At the second, research are being carried out on varied sorts of most cancers, but additionally in primary analysis (e.g. embryonic improvement and plant breeding). Some cell lines are used significantly ceaselessly for this goal.

  • HEK cells , abbreviated for “Human Embryonic Kidney” cells, are sometimes used because of their easy cultivation. They are reworked cells which include overseas DNA from human adenovirus 5. The hypotriploid epithelial cells grow to be adherent and are sometimes used in the improvement of chemotherapy medicine.
  • HeLa cells (named after the affected person Henrietta Lacks) are most likely the most ceaselessly used cell line. In 1951 the first cells have been taken from the line. Since then, working with HeLA cells has revealed over 75,000 scientific articles and 4 Nobel Prizes. The analysis offered findings in most cancers analysis, gene mapping and the functioning of polymerase enzymes.
  • HaCaT cells  are a spontaneously reworked aneuploid, immortal keratinocyte cell line, which originates from grownup human pores and skin. HaCaT cells are recognized for his or her means to distinguish in vitro. The outcomes obtained are extra reproducible.
Multiplexed shotgun genotyping for rapid and efficient genetic mapping.

Multiplexed shotgun genotyping for rapid and efficient genetic mapping.

We current a brand new method to genotyping based mostly on multiplexed shotgun sequencing that may determine recombination breakpoints in numerous people concurrently at a decision enough for most mapping functions, resembling quantitative trait locus (QTL) mapping and mapping of induced mutations.

We first describe a easy library development protocol that makes use of simply 10 ng of genomic DNA per particular person and makes the method accessible to any laboratory with commonplace molecular biology tools. 

Sequencing this library ends in numerous sequence reads extensively distributed throughout the genomes of multiplexed bar-coded people. We develop a Hidden Markov Model to estimate ancestry in any respect genomic areas in all people using these knowledge.

We reveal the utility of the method by mapping a dominant marker allele in D. simulans to inside 105 kb of its true place using 96 F1-backcross people genotyped in a single lane on an Illumina Genome Analyzer.

We additional reveal the utility of our methodology by genetically mapping greater than 400 beforehand unassembled D. simulans contigs to linkage teams and by evaluating the standard of focused introgression strains. At this degree of multiplexing and divergence between strains, our methodology permits estimation of recombination breakpoints to a median of 38-kb intervals. Our evaluation means that increased ranges of multiplexing and/or use of strains with decrease ranges of divergence are practicable.

Multiplexed shotgun genotyping for rapid and efficient genetic mapping.
Multiplexed shotgun genotyping for rapid and efficient genetic mapping.

Direct comparisons of Illumina vs. Roche 454 sequencing applied sciences on the identical microbial neighborhood DNA pattern.

Next-generation sequencing (NGS) is often utilized in metagenomic research of advanced microbial communities however whether or not or not completely different NGS platforms get well the identical range from a pattern and their assembled sequences are of comparable high quality stay unclear.

We in contrast the 2 most continuously used platforms, the Roche 454 FLX Titanium and the Illumina Genome Analyzer (GA) II, on the identical DNA pattern obtained from a posh freshwater planktonic neighborhood. Despite the substantial variations in learn size and sequencing protocols, the platforms supplied a comparable view of the neighborhood sampled. For occasion, derived assemblies overlapped in ~90% of their whole sequences and in situ abundances of genes and genotypes (estimated based mostly on sequence protection) correlated extremely between the 2 platforms (R(2>>0.9). Evaluation of base-call error, frameshift frequency, and contig size instructed that Illumina provided equal, if not higher, assemblies than Roche 454.

The outcomes from metagenomic samples had been additional validated towards DNA samples of eighteen isolate genomes, which confirmed a variety of genome sizes and G+C% content material. We additionally present quantitative estimates of the errors in gene and contig sequences assembled from datasets characterised by completely different ranges of complexity and G+C% content material.

For occasion, we famous that homopolymer-associated, single-base errors affected ~1% of the protein sequences recovered in Illumina contigs of 10× protection and 50% G+C; this frequency elevated to ~3% when non-homopolymer errors had been additionally thought of. Collectively, our outcomes ought to function a helpful sensible information for selecting correct sampling methods and knowledge possessing protocols for future metagenomic research.

A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.

A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.

BACKGROUNDNext generation sequencing (NGS) expertise has revolutionized genomic and genetic analysis.

The tempo of change on this space is fast with three main new sequencing platforms having been launched in 2011: Ion Torrent’s PGM, Pacific Biosciences’ RS and the Illumina MiSeq. Here we examine the outcomes obtained with these platforms to the efficiency of the Illumina HiSeq, the present market chief.

In order to check these platforms, and get ample protection depth to permit significant evaluation, we have now sequenced a set of four microbial genomes with imply GC content material starting from 19.three to 67.7%.

Together, these characterize a complete vary of genome content material. Here we report our evaluation of that sequence knowledge in phrases of protection distribution, bias, GC distribution, variant detection and accuracy.

RESULTSSequence generated by Ion Torrent, MiSeq and Pacific Biosciences applied sciences shows close to good protection behaviour on GC-rich, impartial and reasonably AT-rich genomes, however a profound bias was noticed upon sequencing the extraordinarily AT-rich genome of Plasmodium falciparum on the PGM, leading to no protection for roughly 30% of the genome.

We analysed the flexibility to name variants from every platform and discovered that we may name barely extra variants from Ion Torrent knowledge in comparison with MiSeq knowledge, however on the expense of the next false constructive fee. Variant calling from Pacific Biosciences knowledge was attainable however larger protection depth was required.

Context particular errors have been noticed in each PGM and MiSeq knowledge, however not in that from the Pacific Biosciences platform.CONCLUSIONSAll three quick turnaround sequencers evaluated right here have been capable of generate usable sequence. However there are key variations between the standard of that knowledge and the functions it’s going to help.

A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.
A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.

Compaarison of next-generation sequencing techniques.

With quick improvement and vast functions of next-generation sequencing (NGS) applied sciences, genomic sequence info is inside attain to assist the achievement of targets to decode life mysteries, make higher crops, detect pathogens, and enhance life qualities. 

NGS techniques are sometimes represented by SOLiD/Ion Torrent PGM from Life Sciences, Genome Analyzer/HiSeq 2000/MiSeq from Illumina, and GS FLX Titanium/GS Junior from Roche.

Beijing Genomics Institute (BGI), which possesses the world’s largest sequencing capability, has a number of NGS techniques together with 137 HiSeq 2000, 27 SOLiD, one Ion Torrent PGM, one MiSeq, and one 454 sequencer. We have gathered intensive expertise in pattern dealing with, sequencing, and bioinformatics evaluation.

In this paper, applied sciences of these techniques are reviewed, and first-hand knowledge from intensive expertise is summarized and analyzed to debate the benefits and specifics related to every sequencing system. At final, functions of NGS are summarized.

NGS QC Toolkit: a toolkit for quality control of next generation sequencing data.

NGS QC Toolkit: a toolkit for quality control of next generation sequencing data.

Next generation sequencing (NGS) applied sciences present a high-throughput means to generate great amount of sequence information.

However, quality control (QC) of sequence information generated from these applied sciences is extraordinarily necessary for significant downstream evaluation. Further, extremely environment friendly and quick processing instruments are required to deal with the big quantity of datasets.

Here, we’ve developed an software, NGS QC Toolkit, for quality examine and filtering of high-quality information. This toolkit is a standalone and open supply software freely out there at http://www.nipgr.res.in/ngsqctoolkit.html.

All the instruments within the software have been applied in Perl programming language. The toolkit is comprised of user-friendly instruments for QC of sequencing information generated utilizing Roche 454 and Illumina platforms, and extra instruments to assist QC (sequence format converter and trimming instruments) and evaluation (statistics instruments).

A spread of choices have been supplied to facilitate the QC at user-defined parameters. The toolkit is predicted to be very helpful for the QC of NGS information to facilitate higher downstream evaluation.

NGS QC Toolkit: a toolkit for quality control of next generation sequencing data.
NGS QC Toolkit: a toolkit for quality control of next generation sequencing information.

Preparation of decreased illustration bisulfite sequencing libraries for genome-scale DNA methylation profiling.

Genome-wide mapping of 5-methylcytosine is of broad curiosity to many fields of biology and medication. A spread of strategies have been developed, and several other have not too long ago been superior to genome-wide scale using arrays and next-generation sequencing approaches.

We have beforehand reported decreased illustration bisulfite sequencing (RRBS), a bisulfite-based protocol that enriches CG-rich elements of the genome, thereby reducing the quantity of sequencing required whereas capturing the bulk of promoters and different related genomic areas.

The strategy offers single-nucleotide decision, is very delicate and offers quantitative DNA methylation measurements. This protocol ought to allow any normal molecular biology laboratory to generate RRBS libraries of excessive quality. Briefly, purified genomic DNA is digested by the methylation-insensitive restriction enzyme MspI to generate brief fragments that include CpG dinucleotides on the ends.

After end-repair, A-tailing and ligation to methylated Illumina adapters, the CpG-rich DNA fragments (40-220 bp) are dimension chosen, subjected to bisulfite conversion, PCR amplified and finish sequenced on an Illumina Genome Analyzer. Note that alignment and evaluation of RRBS sequencing reads usually are not coated on this protocol.

The extraordinarily low enter necessities (10-300 ng), the applicability of the protocol to formalin-fixed and paraffin-embedded samples, and the method’s single-nucleotide decision extends RRBS to a extensive range of organic and scientific samples and analysis purposes. The whole course of of RRBS library building takes ∼9 d.


Cytokine storm of Sars-COV2
Cytokine storm

On January 25, 2020,” Prof Bin Cao’s group published a research paper titled: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China . The newspaper demonstrated that the immune system of patients using SARS COV2 (a disorder brought on by the SARS-CoV-2 disease ) is a deadly cytokine storm. So what’s the COVID-19 cytokine storm? Why does COVID-19 result in cytokine storm? And just how to see to the cytokine storm brought on by COVID-19?

What is Cytokine Storm of COVID-19 Cytokine Storm?

Before introducing COVID-19 cytokine storm, then we have to find the understanding of cytokine storm. As described from this article entitled: Everything You need to find out about Cytokine Storm and Virus Infection, cytokine storm is the occurrence which a wide variety of cytokines from the body fluids are quickly and exceptionally produced after your system is infected with germs. This is in fact a high number of viral diseases tripping the “suicide attack” of the individual immune system.

COVID-19 cytokine storm actually denotes the cytokine storm brought on by COVID-19. Some reports have shown the direct cause of death from severe COVID-19 is the novel coronavirus ruin the immune system and activate excess immunity causing cytokine storm. Additionally, cytokine storm damages lungs along with a number of organs of the body (kidney, heart, liver, etc.), and finally resulting in multiple organ acts exhaustion.

Why Does COVID-19 Lead to Cytokine Storm?

The reason for COVID-19 is SARS-CoV-2 (previously called 2019-nCoV), among coronavirus. The SARS-CoV-2 infected people for the first time, and also the immune system did not recognize the virus. Various studies have proven the SARS-CoV-2 moves cells via angiotensin-converting enzyme 2 (ACE2). Because of this, lung tissue has come to be the most important intrusion goal of this SARS-CoV-2 with higher expression of ACE2. Following the virus entered the lung, then the immune system delivered a high number of cells into the lung tissue to kill the virus. This shaped pneumonia, along with the individual revealed cough, fever, and trouble breathing.

Nonetheless, these immune cells can’t track down the virus accurately as they’re not recognizing it. After a cytokine storm is shaped, the immune system might not have the ability to kill the virus, but it is going to surely kill a significant number of cells from the gut, and this will badly harm the role of the lung. Patients may have respiratory failure till they die of hypoxia.

Which are The Cytokines of Cytokine Storm Brought on by COVID-19?

According to the Book of Prof. Bin Cao’s Group, they Found that acute patients have higher rates of plasma pro-inflammatory variables (IL2, IL7, IL-10, GSCF, IP-10, MCP-1, MIP1A, TNF-α) than moderate patients using COVID-19, and such inflammatory signs indicate that a cytokine storm in acute patients.

Acturally, because the outbreak of the SARS outbreak, the “cytokine storm” has sparked great attention. And all them are belong to coronavirus. Even though the analysis of SARS-CoV-2 isn’t really big, we could consult with the analysis of 2002 SARS CoV and MERS CoV. Preliminary studies suggested the cytokine storm brought on by SARS is mostly associated with IL-1β, IL-6, IL12A, IFN-γ, IP10 and MCP1, along with the cytokine storm brought on by MERS are chiefly associated with IFNγ, TNFα, IL15 and IL17A.

Detection of elevated inflammatory elements in blood. And Various viruses Don’t activate cytokine storms through precisely the exact same mechanism, so that they are going to Cause distinct cytokine changes.