Science method

Next Generation Sequencing - Science method

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Questions related to Next Generation Sequencing
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What is the data output for Minion Flow Cell (R10.4.1) without washing and with washing? How many times it is advisable for us to wash the flow cell? Thanks
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Hello Muhamad
We generally also acquire 15–20 Gb per run for long reads (see http://www.jmdjournal.org/article/S1525-1578(23)00194-0/fulltext). This is comparable to using shorter reads in our studies (not published yet). We haven't washed and reused because we generally exhaust the pores/channels.
All the best,
Marcus
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Hello,
I recently received metagenomic 16S rRNA gene sequence data from a company, which includes both raw reads, and clean data with barcodes removed. My goal is to analyze these sequences and obtain information on the taxonomic diversity and abundance of the species present in the sample.
Since I use a Windows system and cannot utilize Mac or Linux, I would greatly appreciate guidance on how to proceed with this analysis. Are there any web server-based applications available that can assist with this task?
Furthermore, if there are any researchers or experts interested in this project, I would be grateful to explore potential collaborations. Please feel free to reach out to me if you are interested or have any recommendations.
Thank you in advance for your assistance.
Best regards
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SilvaNGS - simple and win-based. https://ngs.arb-silva.de/silvangs/ It is probably less flexible than all nice approaches mentioned above but it is quite useful for first look or for someone who needs an established reliable pipeline
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we have already diagnosed 25 cases of DMD ..but unable to perform carrier analysis
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Yes Anjan Da. You can do the screening using different methods like multiplex ligation-dependent probe amplification (MLPA), RFLP, comparative genomic hybridization (CGH). but the best cost-effective strategy would be qPCR followed by sanger sequencing and monitoring CK level. but that will be time-consuming. Again, as the disease is associated with deletion, duplication and small mutation and SNP, NGS would have been a reliable option.
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Which is the best tutorial to learn Next-generation Sequencing online ?
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I have to sequence miRNA from my research plant (common bean), and I got an RIN number between 5 and 6. Does this affect the NGS analysis?
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It depends. Did you extract only the miRNA or the total RNA? If it's a micro RNA specific kit, a low RIN is to be expected, since it will filter out most of the ribosomal RNA that is required for the analysis. If you extracted total RNA, it could mean that your samples are degraded and this could then influence your downstream analysis.
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Currently, I am doing an experiment related to microbiome analysis from environment. However, I face technical problem related to NGS analysis that I have to wait for the analysis for around 2 months, cause the company needs to collect enough sample for one time running. I want to ask is there any faster method to do the microbiome analysis other than NGS?
I read that MALDI-TOF is fast, accurate, and reliable method for microbiome analysis. But from what I read, for this method we need to culture the bacteria and analyze each colony in different running time. Is that correct? If so, then how to analyze uncultured bacteria that might be exist in our sample? because for NGS, we extract the DNA of all types of bacteria including the uncultured one.
Or is there any method to estimate the diversity of bacteria cells in our sample?
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Thank you so much for your brief explanation. Sorry, I might not describe my questions accordingly. I was just curious if we could identify or estimate bacterial composition in my samples without relying on the NGS.
I read that MALDI-TOF is quite reliable for bacterial identification, but it needs a single or pure bacteria colony. So, it would be hard to identify bacteria that are still unculturable. I understand that the majority of bacteria from environmental samples are still uncultured or unculturable.
Anyway, thank you for your insight.
Regards!
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Can anyone tell me about the exact concentration of Agilent SureSelect NGS Panel(RNA probe)?
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Guide RNA (gRNA) plays a crucial role in CRISPR-Cas9 gene editing as it directs the Cas9 enzyme to the target DNA sequence. To improve CRISPR-Cas9 gene editing efficiency, researchers often focus on optimizing the design of gRNAs.
Some strategies for improving CRISPR-Cas9 gene editing using gRNAs include:
1. gRNA Design: Careful selection of gRNA sequences is essential for efficient targeting. Several online tools are available to predict gRNA efficiency and potential off-target effects.
2. Delivery Methods: Choosing the right delivery method, such as viral vectors or lipid nanoparticles, can enhance the delivery of Cas9 and gRNA to the target cells.
3. Chemical Modifications: Adding chemical modifications to gRNAs can increase their stability and reduce off-target effects.
4. Multi-gRNA Approaches: Using multiple gRNAs targeting different sites within the gene of interest can improve gene editing efficiency.
5. Base Editing: Advanced CRISPR technologies like base editing allow for precise changes to specific bases without creating double-strand breaks, potentially reducing unintended mutations.
It's important to note that the CRISPR-Cas9 field is continuously evolving, and researchers are continually exploring new methods to optimize gene editing efficiency and minimize off-target effects. Always refer to the latest scientific literature and protocols for the most up-to-date information on improved CRISPR-Cas9 gene editing techniques.
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To make a comparison between a local strain of bacteria (for example E. coli) with the standard one (NCBI) in terms of genetic content using next-generation sequencing (NGS), what is the lowest number of bacterial samples that must be sequenced to get reliable results?
Thank you in advance for any help.
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If you want to analyze genomic variants and you have a reference genome (as you mentioned in the description), I would do at least two samples each to be sure the variant is something general and not specific (~unique) for the particular sample. This could give you a hint about what you're looking for.
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I want to collect all the available NGS data of a bacterial strain collected from a certain country, how can I do that?
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Consider checking BV-BRC(https://www.bv-brc.org/) and filter data based on your desired location.
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I have been using a set of PaxGene tubes to collect blood samples from which I have been isolating cfDNA for NGS analysis. I need to collect more blood samples, but I noticed that the expiration date of the tubes has passed. I have read some comments on this issue which say that the vacuum of the tubes is lost but the preservation medium does not expire that fast. Does anyone have any experience or information on how good it is to use these tubes for continuing blood collection and whether it would affect the cfDNA yield or quality for the NGS experiments?
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Hello everyone, I want to learn about the recipe for the solution in the cfdna tube, can anyone guide me on this recipe, via email: lap9561@gmail.com
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I have developed this multiplex PCR panel for next generation sequencing library prepration. This panel is used for the diagnosis of a particular bacteria infection, as well as some SNP I'm interested in.
The panel works well with sputum samples, but failed to detected some expected SNPs when we tested with FFPE samples. The copy number of this bacteria might be lower (120) than my detection limit (250). We still managed to get at least 5000 coverage in most of the SNP locations. But only about half the SNPs were called, why not the others?
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amplicons panels need to be sure samples are in good shape and designed amplicons not too long. dis you test quality of your samples (DIN or RIN) and how long are your amplicons?
fred
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I have been using the DNeasy kit from Qiagen to extract DNA and, in the final step of the extraction, the product guide recommends to elute the DNA in AE Buffer that contains EDTA (0.5 mM). However, the Nextera protocol might be seriously affected by EDTA during tagmentation. Could I use TE buffer (0.1mM EDTA) for storing DNA samples in a stable manner for a long time? Or would I have to extract my samples only in nuclease free water for immediate use?
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Hi Moises , I agree with Jatesh , yes you can use EB without any EDTA
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I am currently working in the process of automating our research lab to be able to process a higher number of samples. Our work consists in detecting and quantifying pathogens in mammalian samples (e.g., blood, faeces, tissue, swabs). As such, I am interested in an automated extraction system which produces good DNA/RNA yields to be sent for NGS.
Until now, we have been using Qiagen kits for our manual extractions, so I thought that a machine from the same brand would do for us. However, I've been told that the QIAcube Connect does not really take that much work out of your hands, and that the sample volume obtained at the end of the process with the QIAcube HT is way lower than the one with the manual kit. I have also checked other machines, such as Thermo Scientific's Kingfisher Flex and its kits, but do not know how well they do in comparison with Qiagen's kits.
Based on your experience, which automated extraction system would you recommend? And which brand of kits have you used with it? The system and kits do not need to be from the brands mentioned here (as long as the produce good results).
Thank you very much in advance.
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Laia-M. Pardinilla back when i worked in the diagnostic laboratory, i used CyBio Felix from AnalyticJena. It is also based on magnetic beads technology and it is fully automated, you can modify the program, customize configurations.. and we had two of them, so we could process 192 samples at once, i really liked that one https://www.analytik-jena.com/products/liquid-handling-automation/liquid-handling/flexible-benchtop-liquid-handling/cybio-felix-series/
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Dear all,
Does anyone know the protocol for the best bacterial DNA isolation from raw milk? I would like to perform a full NGS identification of lactic acid bacteria and am looking for the best kit or procedure. Does anyone have experience with such research? Raw milk (brestmilk) hasn't got a large microbiome, hence it is not possible to collect bacteria by microfiltration. I've found a few articles about it, but I'm still looking for researchers who have any experience with such research. Anyone?
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ultrafilterize the raw milk from fats and proteins.
acid precipitation and centrifugation for fats. and special starin for casein proteins. most bacteria should be with the watery part that leavesthe rest of the fats and proteins.
What we used to do is it suspend around 100uL into 900 of buffer or water and then homogenize it.
Afterwards, centrifuge at 10,000G and collect 400uL with the pellet inside.
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Hello,
Can I store whole blood in RNA later and later to use that blood for NGS sequencing?
There is no possibility to stored otherwise and the quantity is very small. Thank you!
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Mojtaba Najafi Thank you very much for your answer! It helps me a lot!
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Due to the constraint of sample, I cannot re-collect the leaves from a genotype that was used for NGS analysis (can only produce draft genome) before. To improve that assembly, can I use the NGS data generated from another genotype (same species but apparently have high heterozygous rate compared to the old one)? If so, please advise for appropriate tool/pipeline and papers dealing with the same issue.
With my appreciation.
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Combining sequence datasets from different genotypes can potentially improve genome assembly quality, especially for regions that are highly variable or difficult to sequence. However, there are several challenges that need to be considered when combining sequence datasets from different genotypes:
  1. Genome complexity: The genome complexity and size can vary significantly among different genotypes, which can affect the quality and completeness of the genome assembly. It is important to ensure that the combined datasets cover the entire genome and do not introduce bias or gaps in the assembly.
  2. Differences in sequence quality: The sequence quality and read length can vary among different datasets, which can affect the accuracy and resolution of the assembly. It is important to carefully evaluate the quality of each dataset and perform quality control measures to minimize errors and artifacts.
  3. Polymorphisms and variations: Different genotypes can have variations, such as SNPs, indels, and structural variants, that can cause challenges in the assembly process. These variations can create complexities in the assembly, such as chimeric contigs, and can also result in incorrect or incomplete assemblies. Special consideration should be given to these variations, and bioinformatic tools such as haplotype phasing and polishing should be used to resolve them.
  4. Computing resources: Combining sequence datasets from different genotypes can increase the computational requirements, especially for large and complex genomes. Sufficient computational resources, such as high-performance computing clusters, are required to process and analyze the datasets efficiently.
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Current application is library quantitation for NGS.
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I used Qubit extensively and think it's easy of use is great and many core centres will recommend Qubit. Quantas looks essentially the same but considering whenever I've used Promega compared to other companies, they usually ended up performing worse (primarily their wizard kits versus the NEB monarch kits), so would go with Qubit over Quantas.
The figures they use are comparing are against the Qubit 3, Qubit 4 has been out a long time it and seems like a dated comparison to look better than they are
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Does anybody have a clue how to analyse targeted RNA NGS data with respect to normalization? How to correct for differences in capturing efficiency per transcript? We isolate patient RNA from PAX gene, perform a Sureselect sample prep (oncopanel) and want to see which genes in a patient are differentially expressed. For whole transcriptome analysis we use the "DROP pipeline".
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I do not recommend following Anita Tripathi advice. It is completely irrelevant to what you are asking (and outdated).
I do not recommend using targeted RNA-Seq of a single transcript for expression estimates. If you must, here are my 2 cents.
Normalization of targeted RNA is complicated if not impossible. Two main reasons: 1) differences in capturing efficiency per transcript and per experiment - unknown unless you do extensive testing every time, 2) variable sequencing depth with no reference. The only solution that comes to my mind is adding spike-in molecules with known expression/concentration and using their levels for the normalization. This would, however, most likely require you to design synthetic spike-in molecules with the same target regions as your transcripts (to estimate capture efficiency) in addition to "classic" spike-in molecules (to have a reference for sequencing depth normalization).
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My undergraduate thesis partner and I will be identifying and characterizing the microbial diversity in 3 sample sites from an urban river. We were thinking of using Sanger sequencing since we would just like to identify what is in the river, but NGS yields a lot of data to really characterize the bacteria present. Our main issue with NGS however would be cost. We are planning to apply for subsidies but our budget is around PHP 20,000 or USD 360. If we will be sending 3 samples, which institution has the most affordable NGS sequencing service? What are possible alternatives to NGS that are more cost-friendly to undergraduate students?
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For Sanger, you need a purified PCR product. I am not sure what you mean by as much data as possible - you will get a single consensus sequence for each direction up to ~800 base. I would recommend talking to a sequencing lab or whoever has a sequencing machine at your university to discuss your options.
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The concentrations of DNA extracted from avian stool samples are extremely low ( about 0.5 ng/ uL, using nanodrop spectrophotometer). May someone suggest me any suggestion to improve my extraction methods using Qiamp Fast DNA Stool Mini Kit for running the NGS to identify the diet analysis using ANML primer set. I have done exactly as the manual provided by the kit but still got low DNA concentration which the highest that I can is about 6.7 ng/uL but A260/A280 is 1.05 and A260/A230 is 0.21.
thank you.
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Péter Gyarmati I strongly recommend using the PCI (Phenol-chloroform-isoamyl alcohol) method as it will give you more DNA than any other method and I strongly recommended using ethanol (70%) for washing.
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It is important for us, that the sample data can be link to the laboratory practices (what is going on with each sample), very good traceability of samples, possible linking to results and analsys of samples, produucing and saving protocols, adding photos, etc.
We are mostly working NGS sequencing (Ion Torrent S5).
Regards,
Aja B.
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If you are looking for something like "best" or "very good" in the context of question, in my opinion, probably you are looking for the wrong thing. You can try LabCollector which offer a trial version, and make your own integration of LabBook and your NGS data and other stuff you mentioned.
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Hi every one
I want to setup a new NGS based kit for my project.
In our project, other researchers used to work with one kit and It had good detection rate
some samples was False negative
The second generation kit is much more comprehensive and I want to set up it
How do you design experiment for the second kit?
Do you start with samples which were proved to be positive?
How do you choose the best minimum Input for library preparation?
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The problem with such questions are that only the person asking question know what the terms used mean. In here NGS, kit experiment and experimental design does not make sense to the reader. Try to ask question with enough details which make sense to the reader and sufficient to get some ideas/answers.
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Current research often uses new, next-generation, "flashy" experimental techniques (i.e. single-cell RNA-seq) that have replaced some of the older, smaller, yet fundamental experimental techniques. Many of these new-age techniques seem overused and expensive when older techniques could be an adequate replacement. What are some good examples of these new "answer-all" techniques and how were these techniques done previously with smaller, fundamental techniques?
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I nominate DNA methylation clocks to measure aging. The problem is not that it's expensive, but that they don't work in the context of intervention. People need to double-check on whatever their methylation clock is saying with a good old life span study. But nobody has time for that anymore, and if they did it, they wouldn't like the result. Instead, they just take the clock on faith.
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  1. A latest Book holding details on every important aspect of Next Generation Sequencing and Genomic Data Analysis with newest available tools
  2. Book with examples in Command Line
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Have a look at this one which I've previously purchased:
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For example NGS should be used to determine types of cancer but I need further examples
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Sanger sequencing is further running of bioinformatics Developed by Dr Sanger's
can be use when you when to sequence limited number of DNA
Template because it is first generation of DNA
It is more expensive, time consuming and not as fast as Next generation DNA sequences
Large numbers of DNA template can be studied and compare at faster and cheaper rate
More bioinformation can be obtained using this second generation of DNA sequences or next generation sequences at a faster and more efficient way
Uses in veterinary medicine
Can be use to monitor origin and spread of disease in a community by analysis DNA template sequences from obtained from public waste
Also further viral mutation or changes in nucleotides can be analyse at faster rate
New or emerging viral disease can easily be detected by using this Next generation sequences
However best and more advanced DNA sequences technique are now available that faster ,more specific,more accurate and cheaper know as third generation DNA sequences technique
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Dear all,
how does it cost a whole genome NGS analysis per sample (150 sample in total)?
Any suggestion on which is the most cost-effective company to do this job?
Thank you.
Oronzo Catalano
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It's not really possible to answer that without more information.
But in general, the price depends on what you sequence (lots to choose from here: is it metagenomics, single-cell, single genome, transcriptomics, ...), and a lot on the amount of bases you want to sequence, which again depends of you samples and your desired coverage.
A way to go about this is to just contact 2-3 sequencing companies, describe what you want to sequence and get quotes from them and compare. Be prepared for 5 figures
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Typically, interaction network analysis uses binary data (absence/presence) or abundance data (frequency) to analyze the network structure (nested or modular). When the abundance data comes from direct observations of the interactions, it makes sense to use them instead of just the binary data because the interaction frequency is biologically important. When one obtains information about the partners involved in an interaction from NGS (for example, fungi associated with plant roots in mycorrhizal interactions), it is usual that some fungi appear more frequently than others (that is, there is a higher frequency of readings of some sequences than others). Does it make sense to use the number of reads as abundance data to build the networks and evaluate their structure, or would it be more prudent to simply use binary data?
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Hello! I am very new to NGS. But It may be good for to learn. Those readings, are same sequences occurring repeatedly occurring repeatedly in a same genome?
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Hi there!
We are doing some tests to sequence different panels in a same FlowCell of NovaSeq 6000. We have already successfully loaded different libraries from different panels in the same FLow Cell. However these panels often produce similarly sized libraries (around 350pb).
Now, we need to include another distinct panel. However, in this time, the panel produces smaller libraries (around 270bp).
We know that different libraries with different sizes show distint clustering efficiency, once smaller libraries cluster more efficiently and probably will be overrepresented compared to larger libraries in a same NGS run.
So, do you have experience in mixture diferents libraries with diferent size to sequencing? Do you have any recommendations regarding the proportions to follow (Considering output per sample, library size, others factors)?
Thank you in advance for your attention regarding this matter.
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Hi,
It is not ideal to pool different libraries (of different sizes) in one run.
But it can be done with some considerations. As you mentioned that small libraries will get overrepresented compared to large ones. Therefore while pooling the libraries you need to make sure you add different libraries accordingly.
For eg:
If you have two libraries:
Lib1- 10 samples and size of 500bp
Lib2- 10 samples and size of 250 bp
and you need equal number of reads for all 20 samples (both libraries)
In this situation, you can not pool both libraries in equal proportion. You need to load the larger library in high proportion as compared to the smaller one.
The exact proportion can be determined by doing a spike run if that is possible.
But if the size difference is less than 100bp then I do not think it will skew drastically.
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Variant genomes may share quite similar sequences, when I make the denovo assembly, only a few contigs are generated (sometimes the variants should contain a large diversity, e.g. HIV). Normally people use SGA for this purpose, want to know how to achieve the same goal using NGS short reads, single genome sequencing such as nanopore should work.
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I think once you get your data (Reads) and you are done with QC, do read clustering. Clusters are formed according to relatedness. You can use ISONClust. Different haplotypes will be generated if present
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In silico testing with Primer Blast and MFE primer 3.1, shows many nonspecific Human amplicons for HPV PGMY09/11 primers. Therefore, are the amplicons feasible for NGS (Illumina, Nanopore) based HPV sequencing?
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Hello, Pushkal Sinduvadi Ramesh, Thank you for your valuable suggestions and the provided links.
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I want to know which type of magnetic beads can be used for pcr cleanup during library preparation of nucleic acid for NGS.
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Generally SPRI / AMPure beads are used for the cleanup, however, it would be better if you follow your protocol or contact your sequencing facility for the correct information.
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I'm looking for a way to duplicate the sequence of a specific chromosome.
I want to do whole genome sequencing and want to get data for each chromosome.
I have been studying the genome of bacteria so far, but I am planning to study the chromosomes of eukaryotes.
However, since there are cases in which chromosomes are shared between closely related species, I would like to conduct research on sequencing by chromosomes to create data for each chromosome and compare them based on that.
The process I am thinking of is as follows.
1. Separation of each chromosome
2. Chromosome-specific gene amplification
3. ngs analysis
4. Production of whole genome data for each chromosome
How can the separated chromosomes be amplified in process 2?
If not, should each chromosome be isolated from multiple cells?
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Hi. You can using NSG and sent all question for specific company , I will write URL for this company , if you like sent question to them (https://genohub.com/)
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I just want to get understand the Ion Torrent NGS platform
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hello
Please introduce me the companies that provide biotechnology services such as designing different types of primers, NGS, RNASeq, etc.
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Following companies have offices in Tehran
Sanofil - Roche - Novo NorDisk - Novartis - Bayer - Johnson & Johnson - Merck KGaA - Zoetis - TCI - BioHorizons Implant Systems. In US many are located in Philadelphia, Pennsylvania and Boston, Mine and San Fransisco, California. - But Iran had made working with US companies nearly impossible. For ease of working outside Iran I'd look to China. In general use caution sharing new ideas with companies you have not worked with in past..
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Hi,
I am about to design a method of sample tracking for whole exome sequencing based on SNP profiles. I am going to perform this tracking method using ARMS-PCR or HRM. Therefore first, I need to design an appropriate primer based on SNPs. Can anyone tell me which web tool would be the best for this aim?
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any of these will give you pretty good result
  • Primer3.
  • Primer3Plus.
  • PrimerQuest.
  • OligoPerfect.
  • PerlPrimer.
  • OLIGO.
  • GenScript Online PCR Primers Designs Tool
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Hello everyone, I am using nanodrop to check the DNA purity. I get a good DNA purity value at 280/260 (above 1.7) but for RNA 280/230 less than 1. I need good DNA samples for NGS. Does this value of RNA matter?
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your DNA purity seems ok, instead of checking your RNA on nanodrop you can either run QUBIT assay or just run on the gel and if you get clear bands its fine to use them for downstream NGS library prep.
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How is it possible to identify random mutations induced by environmental factors in a small cohort by the sanger sequencing method without knowing any specific target genes?
What might be the best and simplest method for this purpose without use NGS?
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I agree entirely with Katie A S Burnette that the genome is just too large and for random changes NGS is absolutely necessary these days.
There are many methods described for mutation detection like SSCP, DGGE ,TGGE, chemical cleavage of mismatch, enzymatic cleavage of mismatch but all of these are limited to fragments of less than 1000 bases and often less than 500 bases so are all suitable for targeted mutation detection is likely gene candidates but inadequate for whole genome screening of any genome greater than 30,000 bases. It has been done with Sanger sequencing but took 13 years and billions of dollars.
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counting bacteria based on CFU or colony forming units is a culture dependent technique.
Next generation sequencing is a culture independent technique
Is qPCR a culture dependent or independent technique?
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Sounds like a test question... No, qPCR measures the amount of bacterial DNA sequences, independent of the bacterial culture(s) from which these sequences were isolated. It is not even relevant if the DNA sequences are from living bacteria or from dead bacterial debris.
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Hello, can you help me please?
I'm working with plants, Jatropha curcas especifically, and i need to analyze some genes. The size of these is from 5 kb to 7 kb, and i need to get their nucleotide sequence. There is already a reference genome, but i only want the to sequence fragments of my interest, so i don't think that NGS is a good option.
By the way, i'm from Mexico.
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Did you mean that you want to sequence the entire gene in one go? I had previously isolated smaller fragments of 5-6 kb genes from Hevea using primers from the reference genome, then used thermostable PCR enzymes designed for longer fragments to amplify the whole gene from genomic DNA. Cloned the long fragment to plasmid DNA and Sanger sequenced the gene with the primers designed for smaller fragments. Alternatively can restrict (using restriction enzymes) the PCR-amplified whole gene fragment, clone to plasmid DNA and Sanger sequence it. Since you have the reference genome, it should be easy to decipher the gene sequence.
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Hi Folks,
I have applied NGS technique for discovering and/or diagnosis the viruses infecting several plants and associated with some insects. However, I have noticed that numerous studies that are similar to mine they applied further step that is sanger sequencing. Can we avoid this step in the future application of NGS?
Thanks in advance
Adnan
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Sanger sequencing is very useful as an internal quality control. Despite the improved accuracy of next-generation sequencing (NGS), it may not necessarily be 100% accurate. So, it is widely accepted that NGS results need to be validated with the gold standard Sanger sequencing technique prior to reporting. Though the costs and turnaround time of this approach are considerable, it is necessary. However, you may avoid this step if the errors can be significantly minimized using proper controls. No technique is 100% accurate and needs to be validated in order to prevent human error and obtain reliable results.
Best.
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Hello everyone
Does anybody know a free accessible workshop on how to do and analyse the NGS result?
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Hi Vahid
You can find smth interesting for you here:
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I am searching for ready available kit or panel solution from any reputed manufacturer for diagnosis coagulation genes by NGS method. We have Genexus and S5 machine from thermo scientific and Novaseq 6000 from Illumina.
Expected gene covered : F10, F11, F12, F13A1, F13B, F2, F5, F7, F8, F9, FGA, FGB, FGG, GGCX, GP1BA, KLKB1, KNG1, LMAN1, MCFD2, PLG, SERPINE1, SERPINF2, VKORC1, VWF
Please suggest compatible panels if anyone is working or have information.
Thank you.
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Hi Jigarkumar
you can try AmpliSeq for Illumina Hematology Research Panel (394 genes)
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I have extracted DNA from human stool by using The QIAamp Power Fecal Pro DNA Kit.
However, I found that most of my purity reading A260/230 were very low. I plan to send my extracted DNA for NGS. Does anyone have any suggestion on how to improve the reading.
Thank you so much.
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Nurul
Did you use right blank/reference? Sorry for asking!
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Need a help! Basically if someone is focusing on liquid biopsy and next generation sequencing.
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Yes
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The sample was extracted by PAXgene kit, However, its measurements on nanodrop is very poor (report is attached). The current quality is not suitable to be applied for next generation sequencing. Are there possible means can be used to increase its quality?
Thanks in advance
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Hello, you can try to run clean up the sample by running through (new) column again, repeating the wash steps. This can potentially increase the purity, but you may lose yield.
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Hello, I got a question with the units of sequencing products. Most of the phylogenetic study that using ddRAD or other NGS method provided the data size of their analyzed dataset using the unit "loci" or "SNP". While for research using Sanger sequencing and PCR, they used "base pairs" or "nts". If I would like to compare the molecular data size between these researches, is there a transforming algorithms to do it? Or could I just simply use these number to compare (e.g. 1400 bps v.s. 7000 SNPs)? Thanks!
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Why do you want to "compare" the size of markers? I think the absolute size of bp is incomparable between a sequence and SNPs among genome, cause all nucleotides in a sequence are linked and not all of them are polymorphic; while SNPs are polymorphic loci scattered on different positions of a genome, which may be less linked to other SNPs. Also, sequence and SNP provide different aspects of evolutionary information, so I think comparing a single sequence to a RAD-seq data could not provide you some useful information
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Hi all,
I'm performing small RNA library for NGS illumina, and I wonder have you tired use SPRI beads to enrich for a small range of length of your library and how does that work?
My desired fragment size is between 175-185bp, I can chose to run a polyacrylamide gel and excise my desired length. But I'm curious, can I just use the SPRI beads to enrich for fragments of 175-185bp length?
Thanks!
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Hi,
We are considering purchasing a Bioanalyser to increase the accuracy of DNA quantification during library preparation for next generation sequencing. However I have heard that some laboratories now use a Tapestation instead.
Given that Tapestations are more expensive, I wondered if anybody had any advice regarding either of these machines and could offer their own recommendations.
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Both the Qubit and QPCR will give a valuable result. However, qPCR still remains the more reliable quantification result. Several kit works. For me..I used the illumina Infinium kit which is like a mastermind containing about 50 different gene. You can check the illumina website for the list of genes contained and if your gene of interest is among..bravo. Then your works goes faster. Used it o your QPCR Machine to check the QC of your sample. Then you wiĺl get a reliable quantification measurement
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Hello,
My group is attempting to decide whether NGS or RT-PCR would be the appropriate method for analysis.
We want to ensure expression of 5 genes is maintained in our two modified cell lines.
Both should express similarly, however one also produces a protein of choice (this is being confirmed via other testing)
We have 3 cells groups.
WT group
Modified 1 (no protein produced)
Modified 2 (protein produced)
Additionally, we don't necessarily want to quantify the level of expression just a confirmation that modified 1 and 2 express these 5 genes similarly and to a greater degree than WT.
Which method would be the most straightforward?
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RT-PCR would be my recommendation. 3 cell lines x 5 genes in triplicate is less than half a 96-well qRT-PCR plate. Preparing 3 libraries for NGS is most likely going to be more expensive, and would still require validation by RT-PCR.
I would only recommend using NGS if you are interested in looking at global changes in gene expression after your experimental manipulations.
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I am designing a custom NGS run and need to determine the sequence coverage. I am using the Illumina Sequence coverage calculator https://support.illumina.com/downloads/sequencing_coverage_calculator.html
How do I determine the genome/ region size?
Additional information:
Human gDNA will be used. I will have 4 amplicons about 250 bases each from 4 different genes.
Any help would be appreciated. Thanks, Nikhil.
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Thanks Luigi Marongiu but I still have not gotten my data yet. I should have worded better. I am just looking to determine the number of samples I will in my run, and for that I will need the genome size or region size.
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Hi everyone, does anyone here have experience extracting fungal DNA from mouse stool and sending it for next generation sequencing? I have continually run into a problem where I extract DNA from stool and submit it for fungal ITS1 sequencing and have gotten poor unusable data back (poor number of reads per sample). I use the qiagen powerfecal pro DNA kit to extract my DNA. Of note when I send the same samples for 16s bacterial sequencing the data looks fine. I ran a gel on my samples using an ITS1-2 primer including a positive control and do not get the expected ~300 bp band. Instead I get several larger bands (see attached). I have been told by our sequencing core that these larger bands would typically be ignored or not get through the flow cell which is why I never get any fungal sequences back. Has anyone run into a problem like this and how did you overcome it?
Thanks!
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HI,
you might try power fecal Pro kit as the extraction of fungal DNA works a lot better. In addition the amount of fungal DNA will be much lower than the amount of bacterial DNA. Further I would check the primer design, annealing temp and general reaction conditions again. Let me know (DM) if you need more background
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I would like to do next generation sequencing on cells sorted based on an intracellular stain. Is there a best practice for how to fix the cells such that my sequencing run is not compromise?
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Did you ever get this to work? I have the same problem and was looking for an answer. I need to fix my cells for FACS but then need to extract the genomic DNA after for a PCR. Thanks!
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I'm looking for primers that target all (or the majority of) viruses and nothing else for PCR. I believe there are primers for some virus families but I haven't found any that would be called universal viral primers. Thanks.
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Hi Ali,
That is impossible because every virus has its own genome.
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I will sequence the micro RNA isolated from Arabidopsis and want to detect the presence (if any) of small RNA which are nor originated from Arabidopsis genome. Please provide me your valuable suggestions for this study. Thanks
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If your are using STAR to align your miRNA you can use the --outSAMunmapped or --outReadsunmapped options to get unmapped reads
Here's the manual for further information:
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Does anyone know a good way of predicting E. coli K-antigen identity from NGS data? It seems like there are lots of options for O and H antigens (e.g. the excellent ECTyper) but none for the K antigen. I'm hoping to type some strains to better understand the host ranges of a phage collection. Any help gratefully received!
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If you have assembled genomes, this should be easily done. The most simple way I could think of right now is similarity based analysis, for example, BLAST.
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Hello,
I am looking for a lab that can process insect DNA samples for me. More precisely, I would like to do hyRAD on my samples (they are not good enough for ddRAD). I am struggling to find one.
Thank you!
Sophie
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Tyler Chafin thank you very much !
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What NGS analysis approaches do you use? Do you use bioinformations? Do you have software solution to do your own analysis? What do you do?
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I recommend to ask question which is logical or make some sense.
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I am doing targeted sequencing on colitis-associated cancer sample, and found some mutations. So I need to validate 3 synonymous mutation with additional samples. Unfortunately, samples are limited and out of 6 samples, I only found the mutation in 1 sample. Is it strong enough? Or do i need to do further validation with sanger? I am also planning to do functional study of that mutation via in vitro.
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If you only have the empty dna sample tube left of your original sample then whole genome amplification will generate large amounts of dna from the small amount coating the bottom of the tube and then you can amplify and sequence. If that is too expensive then just add 10ul of water to the sample that you have left and pcr amplify but add 4 more cycles than usual and you should get enough amplimer to sequence or restriction digest or whatever method that you want to try next
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Hey guys, I was just wondering whether there are any public clusters for evaluation of NGS data like ? Thanks in advance for your comments!
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I have the best experience with the Galaxy cluster of bioinformatics tools https://usegalaxy.eu/ (it has a cutting-edge workflow for nearly all types of sequencing (DNA, RNA, long reads/short reads, metagenomics, epigenomics,...)
Hope this helps
Martin
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Has anyone had luck with NGS library prep for DNA/RNA samples that had low A260/230 ratio due to guanidine thiocyanate? Our samples are made using the AllPrep Qiagen kit without Trizol (used other lysis buffer) and phenol precipitation does not help much. NB, phenol is not the issue.
Thanks.
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If your A260/230 values are 0.8-1.7, do a 1.8X cleanup with RNAXp beads to remove salts. Do a Nanodrop after and your A260/230 value should now be higher. Start with minimum 1000ng RNA -post cleanup RNA will be about 55%-70% (i.e. 550ng-700ng) depending on how much salt contamination is present in starting sample. Resulting RNA can be used for NGS prep.
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NGS has become an integral part of biomedical research, and with the ever-increasing quantity of sequencing data, as cancer biology researchers, it has become increasingly important for us to analyze this data and determine critical gene signatures. However, given the novelty and the overwhelming expansion of resources and online tools in this field, as beginners, it becomes challenging to find suitable resources to learn how to analyze this data. Can anyone with expertise in this field, kindly guide me on this and help me find the right materials, to begin with?
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Before moving on to NGS data analysis, first try to understand what NGS and its different types are.
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I am analyzing my small RNA seq data on Galaxy, I need to remove all rRNA reads from my data. I downloaded a rRNA reference genome for mice and tried mapping with Bowtie2, but it kept failing. Apparently my rRNA reference file had multiple duplicate names. Where can I get a rRNA reference genome from?
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Hi Sanat Bhadsavle , you can download the entire SILVA LSU/SSU rRNA Database (LSU= large subunit rRNA, SSU= small subunit rRNA). For practical reasons, just merge both LSU/SSU FASTA files. Now you can (additionally) isolate all entries that belong to mice. The SILVA entries contain the whole taxonomy in the name.
The next step, map your RNA-Seq data against our SILVA database, I would recommend hisat2 over bowtie2. The resulting SAM or BAM (mapping) file can be filtered to extract all entries that do not map to your reference.
samtools view -f 4 mapping.bam > unmapped.sam
will store all unmapped reads in the unnmapped.sam file
Cheers
Roman
See here for more information:
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Hello!
I isolated some T-cells with some very interesting TCRs from primary cultures. I sent them to Genewiz to get chromium single cell TCR sequencing done, however the sample viability was super low. I sent them 8 more vials so that they could do a dead cell removal and then isolate the live population and perform single cell sequencing on the remaining cells. The sequencing results show that whatever is left over after DCR is most likely another contaminant cell type, not a TCR. I now only have one vial left, so whatever I choose to do next is very critical and essentially has to work the first try.
At this point I dont care about chain pairing, I can piece that back together afterwards by trial and error, I just want to get some data from these cells. I was wondering if anyone knows if I can thaw my cells directly into RNA later and then do either normal NGS or another single cell sequencing method to get any info on the TCR sequences? Should I just amplify the TCR regions on thawing with some kind of primer pool and then send that for NGS? In general, what's the most robust process for getting out TCR information from low viability samples?
Some other notes:
1. I didnt personally do the T cell isolation but my thinking is they were pretty much exhausted at the time of freezing which is why we have viability issues on thawing
2. They were frozen in 10% glycerol + 10% FBS in a Mr Frosty at -80C and then maintained in LN2 and shipped on dry ice.
3. Observed viability is ~30% on thawing however this could just be the contaminant cell population....
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Definitely a fair assessment, Ive made other samples that have similar properties as this, but none of the other samples Ive generated have responded with as much vigor as this sample.
Definitely will invest in generating another panel of T-cells but from what I can tell so far, this sample had a particularly rare phenotype that i may or may not see again in a relatively limited panel size. every once in a while I remember I have this last vial and wonder if I can do anything with it, in the end its always for the birds...
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Hello, I have done WGS of my bacterial strains and got some preprocessed Illumina sequencing files in .fna format. It has a format like this
>1 length=400016 depth=0.86x the sequence
>2 length=323455 depth=1.00x and so on to >102
I want to know how to deal with this .fna file and which analysis to run on it.
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Abhijeet Singh with a big position and seat comes big responsibilities. It would have been great if you provide some guidance rather than demeaning others.
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I extract environmental DNA from a soil sample and sequenced using Illumina's next-generation sequencing technology.
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DEAR Amsale Melkamu
Genovo: De Novo Assembly For Metagenomes, Journal of Computational Biology: a Journal of Computational Molecular Cell Biology 18(3):429DOI:10.1089/CMB.2010.0244.
Meta-IDBA: A de Novo assembler for metagenomic data,DOI:10.1093/bioinformatics/btr216
GOOD LUCK
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- There was no measures taken before storing
- Precious samples; repeating sample acquisition won't be available
- Later NGS application
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Freezing whole blood samples is not recommended as it causes cell damage. One freeze-thaw cycle could be tolerated, if you thaw the samples at 15-30 degree C. Freeze-thaw cycles generally render blood samples unusable for molecular studies as freeze-thaw can lyse cells and these lysed cells could release nucleases. It would have been better if you would have isolated PBMCs from blood within 6-8 hours after blood withdrawal and frozen the cells instead of freezing whole blood.
PBMCs are isolated by density gradient centrifugation, as different components of blood have different densities and can be separated accordingly. The density gradient medium most used is Ficoll or Ficoll-Paque.
To preserve PBMCs at ultra-low temperatures in liquid nitrogen, PBMCs at 5-10 x 10^6 cells/mL are resuspended in freezing medium containing 10% DMSO + 10%FBS + 80%RPMI medium, and are placed inside a freezing container (i.e. Mr. Frosty) at -80°C overnight to allow gradual and even cooling. The following day, samples can be moved to a liquid nitrogen tank for long-term storage. You could later thaw these cells at 37 degree C for further analysis.
Best.
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I read several papers where researchers use several strains of the same species for analysis of recombination or selection pressure however there are few papers in which researchers use the multiple species belonging to one 'Genus', not the multiple strains that belong to one 'Species'. What difference is expected in the result and how it can be interpreted.
I want to know how my pathogenic organism 'Xyz abcde' is evolved (recombination and selection pressure). 5 different strains of 'Xyz abcde' is sequenced while 9 organisms in Genus 'Xyz' is sequenced.
Which dataset I should use to proceed and why? How it will make difference in the analysis?
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The answer is found in this study:
J Virol. 2006 Nov; 80(22): 11124–11140.
Published online 2006 Sep 6. doi: 10.1128/JVI.01076-06
PMCID: PMC1642140
PMID: 16956935
Recombination and Selection in the Evolution of Picornaviruses and Other Mammalian Positive-Stranded RNA Viruses
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A splice site mutation in human RNA at a VAF of ~50% using NGS was detected. When I use the same aliquot of RNA (for contaminating genomic DNA) in Sanger sequencing using primers that amplify this intronic and exon region, the mutation is undetectable. It has been confirmed that there has been no mix-up on the source of RNA and it has been repeated NGS which confirmed this mutation again and Sanger analysis on cDNA has not produced different results. Looking for suggestions for what might be occurring thanks.
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One possibility when Sanger sequencing gives different results is that the change detected is on the same chromosome as a polymorphism which lies under the 3' end of either the pcr primer or the sequencing primer. This means that only one chromosome is being amplified so the pcr is hemizygous and the chromosome that has the change has not amplified or sequenced. One solution is to move the pcr and sequencing primers to different positions not including the original primer positions. If this is human then check snpdb but even if it is not in the database it is worth moving the primers in case you have found a rare polymorphism that is interfering with amplification or sequencing
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Dear all, I am carrying out a project related to the identification of the pathogens (parasites, fungi, bacteria, virus). I have a very large dataset obtained after doing Shotgun Metagenomic Sequencing on multiple faecal samples of animals. All bioinformatic analyses were performed by a company. For identifiation, they blasted all predicted protein sequences with more than 30 amino acids against the reference proteome of UniProt. Then, they only removed those hits with homology below 30% and the final set of UniProt hits and their relative information (including gene name, protein name, GO annotation, pathway, and taxonomic lineage) was sent to us.
After checking the results for the taxonomic annotation, many innacurate species have appeared, so the company suggested to apply filters on the % identity (70%), query coverage (70%) and subject coverage (40%). Nevertheless, they said there are no references to justify these values, and that it is up to us to decide. Based on our previous knowledge on building phylogenetic trees after PCR + Sanger sequencing, we consider that 95% identity would be better, but we do not really know if that applies to this type of research too. Could somebody please enlighten us on which would be the right balance between identity, query coverage, and subject coverage? We do know that it is a trade-off and that many reads will be lost after the filtering, so we want to maximise what we keep whilst still being able to say that what appears on the obtained dataset is true. Thank you very much in advance for your help.
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  • % identity (70%), query coverage (70%) and subject coverage (40%).
>> This is good enough
But I wonder, if you have metagenomics sequence data, you can assemble and bin the data. In this way, you you don't need to use blast or any other analysis manually. The taxonomy will depend on the completeness and contamination on your MAGs/Bins.
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Hello all,
I´m trying to isolate DNA from streptomyces isolate using Kit (GenElute Bacterial Genomic DNA Kit), but I always get sheared/ smeared DNA (see attached figure - 1% agarose gel, 80V, 45min, fresh TAE buffer, 6µl of DNA on gel).
I isolated carefully without vortexing, from fresh culture. For elution I used sterile water.
I need good quality DNA for NGS.
Do you have any suggestion for protocol improvement or recommendation for some other kit please?
Thank you for answers.
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  1. Using DNA direct for gel without any PCR or anything will always give you smear, unless you are using long/high-MW DNA extraction protocol. Thus, the gel above does not make much logic.
  2. DNA shear depends on the cell lysis step and if your protocol incorporate physical and vigorous lysis, the smear is unavoidable.
  3. NGS does not make any sense if not used in appropriate context. And mostly all PCR based and shot-gun sequencing method might work on most DNA extracts unless the quality (bioanalyser/tapestation) is very bad.
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Commercial tools are also fine.
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Before answering the question, I would like to ask you few questions related to the topic
  1. Do you have knowledge and understanding of NGS and how it is literally translated to type/platform in a practical sense
  2. Do you know what an antibody is based on its structural composition?
  3. Do you have knowledge and understanding of what assembly and annotations are and their purpose?
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We are trying to look into mutational landscape of ctdna in serum/plasma on NGS . Any recommendations on kit for isolation. Does isolation kits have any impact on ctdna mutation detection?
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Thank you @ Yangyi Chen. The kits compares the urine cfDNA. We are using serum for cfDNA mutation. Is there will be difference in fragmentation of DNA and quantity in serum samples as compared to Urine? In our scenario 4.0 ml serum collection is not feasible from cancer patients, as the reference study used 4.0 ml of urine as starting material. Increasing the input amount will definitely increases the DNA yield.
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I have treated some of my plasmid samples with RNase A to get rid of their RNA contamination.
I'm going to send the samples for sequencing afterward.
Is it necessary to clean up my samples after RNase treatment?
Does RNase A make any problem in the DNA sequencing process?
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Yes, you should remove the RNase from your sample.
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Hi. Has anyone had any experience with Nextera Flex/Illumina DNA prep with enrichment on FFPE samples?
I'm having some difficulties in obtaining a reasonable quality of sequencing using this protocol.
The problem is obviously related to the degraded input material I'm working with. I spoke with some colleagues who perform enrichment-based sequencing on FFPE extracted DNA, however, none of them was using DNA prep/Nextera flex library preparation pathway.
This library preparation method is extremely convenient, but I'm having doubts if it's optimal for every type of source DNA.
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Muhammad Shakeel - thanks for the suggestion, I didn't think about reducing the tagmentation time. I thought that this parameter was significant with first version of Nextera, and that in Flex/DNA prep it did not matter.
I'm afraid that I might be forced to look for end-repair method instead of nextera, which is so convenient. But the quality of input material is something that I have no control of.
I also considered the FFPE repair kits - is it worth a shot? There were publications proving that it greatly improves the results of microarray analyses. I'm not sure if it will do the job in case of NGS.
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I have done WGS of my three patients samples. And I have done detailed bioinformatics too. So can I publish these data as an individual paper in a good journal?
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This will depend on the question you are trying to answer. For NGS data, it is important that the results are statistically sound. If you are looking for differences in the genome or gene expression between your patients and control group, that should have good statistical support. f you are doing differential expression analysis of genes, make sure you have done so using established differential expression analysis tools, for example.
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The RNA sample will be used in NGS and I am basically searching for protocol that doesn't involve column purification.
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You can either clean-up your RNA using acid phenol/chloroform, followed by precipitation to remove the protein or you can clean the RNA up using magnetic ampure beads from beckman (link below).
The ampure beads are great and mostly used for robotic base NGS library preparation protocols.
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I need the information about the companies in India offering services of environmental DNA for any organism. Please add website links.
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Hi Dinakarsami, I'd be happy to help you with metagenomics and metabarcoding analysis. Pl. drop your message.
Thiru
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I extracted DNA from ffpe tissue, measuring OD by nanodrop, I find bad DNA quality: 260/230 ratio is about 0,3 and 260/280 ration is about 1,3.