Next Generation Sequencing - Science method

Dear Respected Experts,

I hope this message finds you well. I am writing to seek your kind guidance regarding an issue I am encountering. I would be extremely grateful for your assistance.

My research focuses on the Orthoptera species Schistocerca gregaria, which I collected from three locations: Kunming, Xi'an, and Yan'an. I am currently estimating the population distances using FST. In my VCF file, the samples are labeled as S_gregaria_Kunming, S_gregaria_Xi'an, and S_gregaria_Yan'an. I merged the data using the bcftools command.

Following the general guidelines provided at the Population Genomics Workshop 5, I used the vcftools command for FST calculation as shown below:

For my specific dataset, I adapted the command as follows:                                                                                                                                                                                                            vcftools --vcf all_samples.vcf --weir-fst-pop group_kunming.txt --weir-fst-pop group_xian.txt --weir-fst-pop group_yanan.txt --out pop1_vs_2_vs_3_FST

Here, the text files (group_kunming.txt, group_xian.txt, and group_yanan.txt) list the corresponding sample names as they appear in the VCF file. Despite following these steps, I am encountering -nan values in the output, which has left me confused.

Could you kindly provide guidance or suggest potential solutions to resolve this issue? Your help would mean a great deal to me.

Thank you in advance for your time and support.

Best regards,

Almost every Tagmentation library prep protocol has a reaction buffer which contains a high concentration of DMF but I can't find a reason why it's included in the reaction buffer for the Tn5 transposase. The closest I've found is a protocol which claims it's a crowding agent but that claim is unsourced, and I think might be due to a misunderstanding of a statement in DOI: 10.1101/gr.177881.114 where the authors had to replace DMF with a crowding agent in situations where the starting DNA concentration was very low. (Also, DMF has a much lower molecular weight than common crowding agents like PEG.)

Anyone know why it's there? The only thing I've seen DMF used for in biology is as a solvent for reagents with poor water solubility like X-gal. It's really unusual to see it in an enzymatic buffer, especially at concentrations exceeding 10%.

We detected a mutation with a minor allele frequency (MAF) of 5.8% using NGS. However, the fractional abundance measured by ddPCR was significantly lower at 0.468575073 which represents a noticeable discrepancy between the two methods.

From the literature, I understand that a high concordance is generally expected between NGS and ddPCR for variant detection. Could anyone share insights or suggestions on what might be causing this discrepancy or how to approach resolving it?

I have cfDNA samples extracted from different kits, the tapestation has showed some contamination with gDNA, what is the best method to purify the samples from the gDNA, as I want to do NGS running and the gDNA will affect the downstream process. Does any body have a protocol to do so?

I notice a difference between the applications of Qiagen DNA isolation kits: QIAamp DNA kits, DNeasy Blood & Tissue kits, and Gentra Puregene kits. This makes me wonder: Is there a suitable method for DNA isolation for NGS and SNP analysis? Also, some recommend the phenol-chloroform method and others don't, so what is the truth?

Dear colleagues

I have a GeneRead QIACube station which is specified for no longer supported Qiagen NGS GeneReader workflow (emulsion technology). It looks pretty much the same as casual QIACube, but has other worktable and screen. I just can't put a standard reagent tray into the device.

Is it possible to convert GeneRead QIACube (NGS sample preparation) into a QIACube for NA isolation?

Hello Scientists, i am trying to understand the differences between concordant and non-concordant reads in NGS Alignment, can someone explain each one please?

Hi everyone,

I'm trying to perform ATAC-seq without using any commercial kit, but it's been challenging to find a protocol that aligns with this approach. I’d like to ask if anyone has experience with this.

Here’s the workflow I followed:

ME A: 5'-(index sequence)AGATGTGTATAAGAGACAG-3' ME B: 5'-(other index sequence)AGATGTGTATAAGAGACAG-3' ME rev: 5'-pCTGTCTCTTATACACATCT-3' By following the standard primer annealing protocol, I obtained two transposon oligos (ME A-rev, ME B-rev).

2. Nuclei Extraction: I followed the Kaestner Lab Omni ATAC protocol (file attached) and confirmed the extraction by performing MNase digestion. This step seems to be working fine.

I've attached the protocol for the Tn5 transposase I used. Following this protocol and the Omni ATAC protocol, I set up the transposition reaction with the following conditions:1 µl 10X Tn5 Transposase Buffer (final conc. 1X) 1 µl Annealed Transposon A (final conc. 1 µM) 1 µl Annealed Transposon B (final conc. 1 µM) 0.1 µl 10% Tween-20 0.1 µl 1% Digitonin 1 µl Tn5 transposase Add D.W. to 10 µl I added this reaction mix to the extracted nuclei and incubated at 37°C for 2 hours.

4. DNA Purification: I purified the DNA using the QIAquick PCR purification kit.

After these steps, I performed PCR for amplification and confirmed the product with gel electrophoresis, looking for bands at 300, 450, and 600 bp (corresponding to mono-, di-, and trinucleosomes, with ~150 bp added for index and adapter sequences).

This method worked once, but I haven't been able to replicate the results since then. Instead of the expected bands, I could only observe smears or odd bands after PCR (figures attached). I would really appreciate any advice or insights you might have.

Sincerely, Hyelin

I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using original DNA plasmid as template for PCR. The PCR product contains 02 kinds of DNA which are in the same length (original template and newly synthesized product). PCR product will be treated by DpnI to digest all the original DNA. The remain PCR product (my target DNA, linearized structure) will be purified and performed in-fusion to circularized into plasmid, then transformed to E. coli for propagation. Plasmid will be extracted from the E. coli and confirmed by NGS. I repeat some experiments, unfortunately, it seems original DNA was still partly remain after DpnI or the site-directed mutagenesis reaction was not successful (I think) making new plasmid still identical to the original template.

Now I want to check whether my target amino acid is fixed or not before sending sample to NGS optimizing cost benefit.

while conducting a research with a small sample size (5-6) with a large exome size (21-25), should you go for Sanger sequencing or NGS? Keeping cost, labour and practicality in context.

Hello researchers,

Sorry for my stupid question. I am learning the QIIME2 workflow for analyzing some 16s amplicon NGS fastq data. I found a very nice paper with data and code public available ( ) so I decided to reproduce their qiime2 results.

In their steps, they cut off the barcode and primer sequences from the raw fastq sequences (Yes, there are really barcode & primer sequences at the front of sequences). However, I did not find any primer sequences in my own NGS fastq files. Does this mean that the sequencing company have removed barcodes, adapters and primer sequences for me?

Also, should I perform quality-control before importing my fastq raw data to QIIME2?

I have DNA samples (extracted by using Qiagen kiit), the 260/280 ratio is consistence 2.1.. for most of the samples. Can I proceed for the whole genome sequencing with these values?

NGS.

I have been preparing NGS Library, where the samples input volumes, conditions followed and the PCR Cycles are same but still the concentration obtained was uneven and the fragments size where also differ from sample to sample. What could be the possible reason for this uneven results.

Hi,

I am very new to Bioinformatics. Recently, I have the project that aims to perform taxonomic anlysis on raw reads from mixed samples taken from environment.

For example, we perfromed NGS (whole genome) on a insect and we want to identify the taxonomic of every symbiotic bacteria in the raw reads.

Currently, I can use Kraken2 to perform the analysis. However, I have following few questions.

1. How can I focus only on the bacteria and remove the rest of data and make a summary table or visualization (the percentage of each bacteria strain).

2. Because in the future, we will switch to the mixed environmental samples and focused on 16s rRNA, I would like to know how to perform the taxonomic analysis by identifying 16s rRNA first from the raw reads and make analysis that focuses only on 16s rRNA. Because mixed environmental samples will contain not only bacteria but also other eukaryotic DNA reads, I want to identify them and analyze them later to reduce the process time.

Followed by that, what process and tools shoud I use?

I found the tutorial: " 16S Microbial Analysis with mothur (with galaxy)" however, I tried with the current data of NGS data on insects, it took so much time on making contigs of non bacterial reads. I am wandering if there is any methods that can get rid of reads that is non bacterial in the first hand.

Additionally, I found other tools such as RNAmmer, barrnap, prokka. However, these tools seems to be only accepting bacterial whole genome but not mixed reads.

If you can share some experience and good workflow or tools to try, I will very appreiate that.

Thank you very much for your great help.

Why WES and RNAseg analyses of the primary lung cancer tissue give different percentage of VAF for the same gene?

Nan-Haw

Hello,

Since a large part of my budget will go on NGS and I don't have the money to repeat it, I want to make sure that it will work.

The PCR products look good on the gel (nice bands of the expected length), but I was also wondering whether it's feasible to send a few samples for Sanger Sequencing to verify the product, before I spend all my money of an Illumina run.

Thanks :)

Dear Team Fungi,

We would like to identify root fungi from Vitis vinefera via metabarcoding. The methodology is established, we have had good results with other root samples using the isolation kit 'innuPREP DNA/RNA MiniKit' and the standard primers gITS7ngs/ITS4ngs with 49 °C annealing temperature. Unfortunately, we do not get any bands from Vitis roots, no matter what we try (e.g, adding BSA or a temperature gradient). Does anyone have any ideas on how to modify the DNA isolation or PCR to eliminate the potential interfering substance in Vitis roots? There must be something in there that interferes with the PCR...

With desperate regards

Kai

Greetings of the day,

Dear Friends,

I hope you are doing well. Kindly discuss the general pipeline/methodology/steps to do Next-generation sequencing (NGS). Can Anyone tell me where i can learn the steps?

Please share your expertise regarding NGS.

I thank you in anticipation.

I'm working on library prep for ITS NGS using Earth Microbiome Protocol and am getting double banding and smearing on my gels. What might be the cause for this? I should be seeing a band around 230 bp.

Actually I want to perform RNA seq analysis via NGS platform to identify the genomic variability and diversity in specific viral strains

Dear All,

In my lab, we have performed whole-exome sequencing of 10 patients to see if we found any germline variations associated with prostate cancer.

I am new to projects that use sequencing and am a little lost on how to analyze these results.

Can anyone suggest to me an app, approach, or pipeline to analyze these results?

I have my data in fastq files and understand a little bit of R.

Thanks a lot

Can someone recommend and method/way to calculate a proper number of cells for NGS? E.g. let's say we know that in the experiment we have 1 out of 2000 with a variant, and it is not present in the control. How many cells need to be taken for single cell NGS to detect the variant with 95% CI?

Are there regulatory requirements for the design and organization of rooms for clinical NGS laboratory?

Hello!

I have performed a phiX validation run with Illumina standard phiX kit and V3 chemistry. I diluted the phiX library to 10 pM and expected to get a cluster density of around 1000, but it resulted in a very low amount of cluster density (~130). Also as you can see in SAV plots, phred scores have decreased after cycle 100 in read 1 and cycle 40 in read 2, which resulted in a low >Q30 percentage at the end of the run. What do you think has caused this issue and how I can fix it? Can this be because of the low cluster density? can this be because of bad reagent storage conditions or handling? I have performed a system check and it was successful. what are your suggestions?

I have attached plots of SAV analysis and thumbnail images in different cycles of the run. the photo with better quality is for cycle #17 and the photo with lower quality is for cycle #436, both for A nucleotide.

Thank you.

I am trying to adapt a DNA extraction protocol to allow me to extract both RNA and DNA from the same sample. I plan to put the sample through Qiagen Allprep DNA/RNA mini kit, but I am not sure at what step I lose the RNA.

SDS/CTAB cleanup

a. Add 10 μl SDS (10%) + 1 μl proteinase K (10 mg/ml stock) to each tube and incubate at 56 °C for 20 min. At this point, pre-incubate CTAB/NaCl solution at 65 °C.

b. Add 35 μl NaCl (5 M) + 28.1 μl CTAB/NaCl (2.5%). Pulse vortex. Incubate at 65 °C for 10 min then perform a quick spin.

c. Add 200 μl phenol:chloroform:isoamyl alcohol (25:24:1) pH 8.0. Pulse vortex. Centrifuge 8,000 × g for 5 min at RT.

d. Collect the aqueous fraction. Add 200 μl chloroform. Pulse vortex for 3–5 sec. Centrifuge 8,000 × g for 5 min at RT.

e. Collect the aqueous fraction. This is final Virus Nucleic Acid.

The final viral nucleic acid goes through the Qiagen DNeasy Blood and Tissue kit.

I am not sure if the final nucleic acid contains RNA. If it does not contain RNA, I would like to know at which step I should put my sample through the kit.

I read protocols using CTAB to extract RNA as well as DNA, but I am not so sure about phenol:chloroform:isoamyl alcohol or plain chloroform. I read a similar protocol for extracting RNA that used CTAB and phenol:chloroform:isoamyl alcohol, but they replaced the chloroform with isopropanol and centrifuged, collecting the pellet as final RNA.

If someone could help me sort this out, it would be great!

FYI, this is part of a protocol to enrich for viral particles and extract the nucleic acid from stool with the least amount of human or bacterial contamination.

I am looking for opinions/recommendations of NGS Platforms (Illumina vs. BGI/MGI) from people that had opportunity to work on both hardware: MGISEQ-T7 and NovaSeq 6000 or analyze data from both. If you could please compare both and provide information, basing on your personal experience, on data quality, quality of service/tech support, usability, opinions, problem situations, pros & cons for each hardware I would be grateful.

We are currently working on a project involving the extraction of DNA and RNA from various types of animal samples, such as whole blood, serum, faeces, etc. The objective is to detect various pathogens through Next-Generation Sequencing (NGS). Our approach for each animal is to combine all the extracted DNA and RNA (converted to cDNA) together (e.g., its DNA from faeces + RNA from faeces + RNA from serum + ...), thus reducing the number of samples to be processed during library preparation.

We have an extraction kit that allows us to either extract DNA and RNA together in a single tube, or extract DNA in one tube and RNA in a different tube. Since our intention is to mix them anyway, we are considering the former option. Nevertheless, we are uncertain whether this will impact the RNA-to-cDNA conversion. Will the presence of DNA affect the conversion process? Additionally, are there any potential effects on the integrity of the DNA? While extracting DNA and RNA together would offer significant benefits in terms of saving time, consumables, and reagents, we will not proceed with this option if it might adversely affect the quality of our extracted DNA or RNA.

Thank you very much for your time and assistance.

Regarding interpretation of sequence variants using ACMG rules.

Are ACMG rules PS3 and PP3 exclusionary?

In other words, if both PS3 and PP3 rules are fulfilled, can PP3 can be applied?

PS3 - Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

PP3 - Multiple lines of computational evidence support a deleterious effect on the gene or gene product -conservation, evolutionary, splicing impact, etc.

P.S - For me, they are not exclusionary.

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

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

we have already diagnosed 25 cases of DMD ..but unable to perform carrier analysis

Which is the best tutorial to learn Next-generation Sequencing online ?

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?

Can anyone tell me about the exact concentration of Agilent SureSelect NGS Panel(RNA probe)?

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.

I got DNA as 500 to 2300 ng/ul with 1.5 260/280 ratio but still the color of the DNA is brown , how to remove this brown color??

I need this DNA for NGS analysis whether this is good for NGS analysis ??

I want to collect all the available NGS data of a bacterial strain collected from a certain country, how can I do that?

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?

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?

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?

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.

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?

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!

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.

Current application is library quantitation for NGS.

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".

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?

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.

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.

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?

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?

For example NGS should be used to determine types of cancer but I need further examples

For RNA profiling of frozen tissues, researchers recommend to use single-nuclei RNA sequencing instead of single-cell. What is the reason for this?

Also, what is the best way to freeze cells for RNAseq at a later time?

Thank you very much for your help, be safe!

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

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?

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.

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?

I want to know which type of magnetic beads can be used for pcr cleanup during library preparation of nucleic acid for NGS.

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?

I just want to get understand the Ion Torrent NGS platform

hello

Please introduce me the companies that provide biotechnology services such as designing different types of primers, NGS, RNASeq, etc.

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?

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?

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?

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.

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

Hello everyone

Does anybody know a free accessible workshop on how to do and analyse the NGS result?

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.

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.

Need a help! Basically if someone is focusing on liquid biopsy and next generation sequencing.

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