Next Generation Sequencing - Science method
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Questions related to Next Generation Sequencing
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.
we have already diagnosed 25 cases of DMD ..but unable to perform carrier analysis
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?
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 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.
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?
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.
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.
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).
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?
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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?
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.
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.
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 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?
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.
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
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.
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.
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
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!
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?
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.
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.
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?
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?
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.
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?
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?
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.
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!
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.
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.
Hey guys, I was just wondering whether there are any public clusters for evaluation of NGS data like
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.
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?
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?
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....
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.
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?
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.
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.
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.
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?
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?
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.
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?
The RNA sample will be used in NGS and I am basically searching for protocol that doesn't involve column purification.
I need the information about the companies in India offering services of environmental DNA for any organism. Please add website links.
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.