Next Generation Sequencing - Science method
Explore the latest questions and answers in Next Generation Sequencing, and find Next Generation Sequencing experts.
Questions related to Next Generation Sequencing
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.
Is it suitabale for NGS or i should repeat the extraction
My background is Biotechnology and for my PhD research Bioinformatics (NGS Analysis) which is necessary not easy task.
now days I am learning Linux Kernel (Operating system), which is hard to understand.
as I have MacBook Air, now the question is that can I do All the NGS analysis using MacBook as Mac have Terminal command.
rather learning Linux kernel, because for Linux I need to buy another Machine/ time to learn all the necessary commands.
I want to do NGS for my samples. I sent the samples to the company - they passed the bioanalyzer QC (which means the RNA conc. is enough) but they didnt pass the library QC. what could become the main issue during the process? Thankyou...
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
We would like to perform some amplicon sequencing. Paired-end reads 2x300 or 2x250 bp on miseq system. We are looking for a reliable, reasonably priced and fast sequencing service. Our purpose is to get at least 100 k reads per amplicon.
I have some cleaned degradome-seq files, FASTA format, provided by a company. I found out the length of reads in FASTA files are variable between 45-47nt. I assumed they might contain adapter remnants, so I used Trimmomatic "java -jar trimmomatic-0.39.jar SE -phred33 X1-clean.fq.gz outpute.fq.gz ILLUMINACLIP:TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:15". But, all reads were removed. Could the length of degradome-seq reads be more than 25nt? The attached image shows the quality of raw data.
I am working on cell culture medium, using Ultracentrifuge to collect EVs, and check for size and concentration with NTA.
Is there a concentration (particle/ml) for a miRNA NGS analysis?
There are few articles suggest, after Trizol and Nanodrop checking, some say they put 10ng RNA but some say they put 100ng RNA?
Does any one has an experience on this?
I would appreciate some insight, thanks so much.
I am working with a parasitic nematode. We performed a differential expression analysis based on a reference transcriptome and found some interesting genes. Now, I want to look at the alignment of these interesting genes. I think that should be stored in the BAM files that originated after the mapping. How to extract only the alignments (or mapping) for these genes?. I have searched for tools, but they suggest using an annotation file to look for the genes according to the coordinates. However, all I have is reference transcriptome in fasta format.
Thanks for any suggestion!!
Which NGS method would be most suitable to achieve the following objectives in plant sample
• Identifying all the genes associated with the biosynthesis of a particular metabolite.
• To locate the chromosome regions associated with the biosynthesis of the same metabolite.
This is my first time of collecting water samples from marine environment. Could somebody please tell me the technique? I mean what kind of equipments (I usually used plankton net for freshwater) and procedures, in terms of collecting samples for metagenomic analyses. I could read from articles (and I did) but I need more practical quidance. Thanks a lot!
If the results of DNA sequencing via NGS panel through Ion torrent , are showing variant allele frequencies of the studied genes around 11-14 % on IGV in many patients of the studied cohort, instead of the expected 50% in a dominant disorder. Could we consider this variants to be real or this is probably an artifact ?
N.B Samples are tissue ( parafin sections ), so could this be a sampling issue ?
is it possible to submit or it require other editing or software to get accession number give a suggestion or help me to submit the sequences.
Thanks and regards
Next Generation Sequencing is a quantitative specialized technology that allows the analysis of genes through parallel sequencing of thousands to millions of DNA molecules in relatively short period of time with high-throughput, higher speed, sensitivity, reduced cost, and permitting a discrete discrimination between homozygous, heterozygous and subclonal events based on the number of DNA molecules supporting each variant (allele burden).
We are searching for the most effective technique for analyse Human Leukocyte antigens types
I wanted to know what kind of studies like pipelines, R code, Python code, and software can be used to perform Next-generation sequencing (NGS) specifically for HLA typing. What kind of studies can be performed on HLA data?
I am looking for ideal configuration details for a workstation to perform a metagenomic, transcriptomic and whole genomic analysis.
I have installed the mixcr file in the Home PATH of the Ubuntu from the respective GitHub and tried to set the mixcr directory as an environment variable by using the "PATH=$Home/<directory name>:$PATH". Though the above command did not show any error but am unable to get the access of mixcr function and instead it's showing "Command 'mixcr' not found", so is there any other method to set environment variables?
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.
Is anyone using the same DNA extraction kit for faecal, cecal and oral mouse samples? Please share your experience about the yield, use in downstream analysis such as NGS etc.? I really appreciate any help you can provide.
I recently performed a sequencing run with MiniSeq. The experiment involves editing via viral transduction, where the construct contains the CAs9 and guide RNA. When performing the NGS analysis, the total reads seem to be good enough (good enough depth). However, the number of reads with the indicator sequences in the given amplicon seems to be awfully low compared to the total reads. I bought new primer sequences for NGS prep and repeated the NGS run. No contamination or primer dimer in the library prep. Still I see the same low reads with indicator sequences in the given amplicon.
I think the transduction and random integration can be the reason for imperfect alignment of the amplicon, resulting in low number of reads with indicator sequences. Am I right? Could there be any other reason ?
The analysis was done via Rgen Cas9 analyzer website link http://www.rgenome.net/cas-analyzer/#!
Our research group plans to buy a NGS sequencer . The obvious choice becomes Illumina MiSeq. However, the steep price tag at 1.5 crore is a hinderance. Can you suggest any alternative along with its price with a mention of the drawbacks when compared to MiSeq. Would really appreciate the help. Thank You in Advance.
I am looking to upgrade the GPU in our computer to run real-time basecalling with the MinION MK1C. The recommended GPU's are a little out of our price range so I was hoping to get some idea of what people are using.
Our computer specifications are currently:
ASUS X99-e WS motherboard with Intel Xeon E5.
NVidia Quadro K2200.
I was thinking an Nvidia Quadro RTX 5000
Any advice would be appreciated.
I am trying to sequence the genome of one protist. Apparently, it has some secrets in its RNA world:) In particular, I cannot remove RNA from gDNA extraction (phenol method or salt method). I tried RNase A (30 min to 4 hrs at 37C or OvN at RT, or combination of both). I also tried the mix of RNase A, Rnase H, and RNase T1 (all we had in the lab). Still, I end up with as much RNA as DNA in my sample.
I figured it might be because of some specific folding of this organisms' RNA species?
In this case, are there any other commercially available RNases I can try?
Or can there be another explanation?
It seems that this RNA contamination affects the downstream sequencing (nanopore), so I'm anxious about finding solution:)
Thank you in advance!
I have sorted IgA coated bacterial cells by flow cytometry. The cells are in PBS and stored at -80. The total number of events is 50,000. Since it is a small cell numbers, is there any effective protocol to extract DNA from IgA coated bacteria to run 16S NGS? Originally the bacterial cells are extracted from Mice fecal sample.
Any help would be highly appriciated.
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!
What is the minimum DNA concentration (ng/ul), yield (ug), and purity (absorbance A260/A280 and A260/A230) accepted for Next-generation sequencing (Illumina and Ion Torrent)?
I have a question regarding gene prediction for long metagenomic reads (MinION nanopore).
I was trying to understand the process of gene prediction. In my attempt, I classified my metagenomic sequences using a reference database by following methods :
1. I did Prodigal to predict ORFs using -p meta option and then ran a diamond aligner using e= 0.002
Result: 689 queries aligned
2. I directly used diamond for alignment of metagenomic reads to the reference database using the same e score value (I did not give identity parameter)
Result: 7292 queries aligned
3. I converted my DNA fasta file to protein sequence using GOTRANSEQ, and did the same analysis with same parameter.
Result: 169 queries aligned.
There is a huge difference between 2 and 3 method? Confused...!!
Which approach is better for predicting protein gene sequence for long reads ?
Is e-value a sufficient parameter for diamond blastp analysis? Do I need to give any identity % in case of first approach?
In addition, I would also like to confirm, whether I can directly use the translated file from PRODIGAL analysis (-a output) for DIAMOND?
Does anyone have experience with cfDNA NGS?
We would like to sequence some samples, but I'm currently struggling to find the correct NGS protocol.
We can choose from several readmodes:
- NextSeq550High (300-400million reads)
- NextSeq550Medium (110-130million reads)
-NextSeq2000P2 (300-400million reads)
- NovaSeqSP (650-800million reads per lane)
Which one should we choose?
Further, should it be paired-end or single read?
Last question: I have read that some kits (i.e. QIAamp circulating nucleic acid kit) contain carrier RNA complicating NGS library prep. Should we use a kit without carrier RNA (i.e. Norgen's cell-free DNA preservative tubes dx) just to be safe?
Thank you :)
I have received the annotated data for NGS WGS and we have done some analysis like sequence similarity distribution among species, score distribution of the biological process, etc using Omicsbox. Then we thought of comparing the complete sets of protein and gene (more than 5000 in Fasta format) with protein and gene sets from few other species. Is it possible and how it can be carried out.
I am new to bioinformatics, kindly guide me
Illumina provides a list of primers to amplify with high taxonomic coverage the ITS1 region for further fungal sequencing, but I cannot find the exact amount necessary of each primer. I understand then that have to mix them equimolarly, am I right? Has anyone used them?
I am trying to apply meachine learning approaches such as: support vector machines, random forests, artificial neural networks on a microRNA dataset (NGS) and also on a mRNA dataset (NGS).I have used sequencing data for training and to make classifications based on some features and then tested on an independent data set to see the prediction accuracy.
To calculate support vector machines (SVM) I used the R e1071 package but I am looking for other tools.
Any body can share/suggest an R/ Python code/Package?
We collected faecal samples from various species that we want to analyse their microbial communities' DNA via Next-Generation Sequencing. The problem is that due to budgetary constraints, we will have to pool the isolated DNA from each sample to be able to analyse them all. We will of course keep each species separated from the other ones, and we are planning to pool the samples from individuals which share enclosure only. Nevertheless, we do not know if there is a maximum number of samples that can be pooled together for NGS.
I am a research tech in a wildlife genetics group and my PI and I are trying to figure out how/where to put raw reads in a repository. We have capture-seq NGS from Rapid Genomics. The post-doc who was working on a project left our lab group and has not been in good communication to assist. I am new to working with NGS data, slowly trying to figure it out! Any input would be very appreciated!
Do you recommend storing DNA and plasma aliquots (samples) at -20°C or -80°C for a period of 5 to 7 months knowing that subsequent analysis will be NGS?
Looking expert opinion...
I have collected marine sponge samples and were shadow dried for two weeks. Now the sponge samples are well dried and can be directly powdered by grinding. I would like to study the sponge associated actinobacterial populations (uncultured) from the dried sample rather than fresh sample.
Here comes my doubt,
If we grind and use the sponge powder for metagenomic DNA extraction, does the DNA be damaged/sheared ?
can directly use the dried sponge material (without grinding) for metagenomic DNA extraction?
Kindly, some one clarify my doubts.
Thanks in Advance,
I would like to barcode fungal caps from the field for two main research goals (1) molecular iD of the fungal specimen and (2) to identify any invertebrate DNA within the cap using CO1 primers.
Does anyone have any experience with the versaility of Whatman FTA saver cards (i.e. use with fruiting fungi) and whether it would provide sufficient DNA for the amplification of metazoan communties using Illumina NGS technology?
I need good quality and quantity of viral cDNA for NGS analysis. Please suggest how can I increase amount of cDNA obtained from viral RNA.