Genome Sequencing - Science method

Hello all,

I would like to ask you if anyone has experienced to use more than one year old sample for whole mitochondrial genome sequencing using Nanopore.

How old is too old for ONT sample?

For researchers in molecular biology and genetic engineering, I would like to know the most successful genome sequencing technique(s) currently used worldwide?Thanks in advance.

I am a PhD student, currently studying yeast endobacteria. The endobacteria our group work with are however cultivable although their genome sizes are significantly cut to half. The genome sequences of those endobacteria include some gaps. How to understand whether this reduction in genome size is due to gaps obtained while sequencing or evolution? Maybe this is a naive question but I will be really happy if you guide me through this.

Thanking you in advance.

Sincerely,

Trina Roychoudhury

I want to know the completeness and contamination level of bacterial genome sequence. I have the assembled FASTA file of the bacterial genome sequence. Are there any online tools where I can upload my FASTA file and directly can find the completeness and contamination level?

I have extracted historic DNA from animal tissue using the Qiagen DNeasy kit and eluted the DNA in the provided elution buffer, which contains EDTA. I will perform whole genome sequencing on an Illumina platform. Because of the low DNA yield, I will utilize the TrueSeq Nano or Microplex prep kit. These library prep kits are sensitive to EDTA. I must therefore cleanup the extracts and use an EDTA-free elution buffer. What approach should I use for the cleanup? 

I will avoid AMPure cleanup due to the small peak sizes (<150 bp) in my DNA templates. Grateful for all suggestions!  

I want to know the completeness and contamination level of bacterial genome sequence. I have the assembled FASTA file of the bacterial genome sequence.

Are there any online tools where I can upload my FASTA file and directly can find the completeness and contamination level?

Thanks in advance

I want to know the completeness and contamination level of bacterial genome sequence. I have the assembled FASTA file of the bacterial genome sequence.

Are there any online tools where I can upload my FASTA file and directly can find the completeness and contamination level?

Thanks in advance

I am trying to map Illumina data to rotavirus genome. But I noticed that mapping to different reference genomes results in some variations in the consensus sequence. Any suggestion? Should I discard genome regions with low coverage? If yes, then what is considered low coverage? 

Hi everyone,

I am a PhD student exploring various genome sequencing approaches and NGS platforms before settling on one for my research. While searching WGS info, I found nothing helpful about the computational resources (hardware, software)required for WGS analysis so reaching out to the RG community if someone can share their experience, I'd be grateful to you. Thanks.

Hello,

I am working on viral metagenomics and have started using the High Throughput Sequencing reads for virus genome assembly. Since, I am at the beginner level, I have some confusions. As these are metagenomics samples with unknown viruses, I run de novo assembly of the sequencing reads for getting contigs followed by reference mapping of the reads against the longest contig first with iterative assembly to extend contigs at the ends in Geneious. After that, the extended contigs are aligned with the closest reference sequence for final genome assembly. My confusion is- how can I make it sure that the contig extension was performed correctly (no missassembly). Also, when it comes about visual inspection of contig vs reference sequence alignment, what are the things I need to pay attention for getting a reliable genome assembly? I aligned an extended contig to the closely related reference sequence and it seems that the contig covers only 8-10% of the genome sequence and there are early stop codons in the contig which does not make sense to me as blastn search showed 60% nucleotide identity of the contig with the reference sequence before contig extension. I am under the impression that there is something wrong in the contig extension step. Can you please share your experience of genome assembly using both de novo contigs and reference mapping? Thanks a lot for your time and help!

Imagine you have a whole genome shotgun (WGS) sequence of a bacterium. You want to identify what species it is, based on this genome sequence, but various softwares give different results. Which software would you trust the most and why ? Preferably, choose between RAST, PATRIC-BRC, KmerFinder and TYGS, please.

I would like to sequence bovine mitochondrial genome. Please let me know which sample( blood or other tissues) is best to isolate mitochondrial genes. 

We have obtained partial sequence data from a bacteria and there are assembled contigs of around 24000 nucleotides.

How can I find all the genes coded on both strands of DNA? I use UGENE and found many orfs in there but searching one by one what are they is too much. Is there an easy way to find and annotate all the protein coding regions automatically?

Thanks

Dear all,

In my current project I have to analyze the whole genome of a bacteria. Oxford nanopore MinION sequencing platform was used to get the genome sequence.

Please guide me on the protocol to analyze the raw data and extract mean information.

Thanks a lot in advance

Greetings everyone.

During my master's research, I focused on exploring the potential of a specific bacterial strain to produce antibacterial compounds. To achieve this, I used the technique of liquid-liquid fractionation (Extraction) using butanol for the bacterial culture broth. Then, I subjected the supernatant to freeze-drying and further dissolved a portion of the butanol crude extract in methanol for analysis using GC-MS. The results revealed the presence of two secondary metabolite compounds, notably beta-carboline and cyclo-l-proline-l-leucine.

I investigated the genes and enzymes of the bacteria, and it appears that the genes and enzymes that synthesize beta-carboline and related compounds were not present in the bacteria. I have the genomic sequence date of the bacteria. I have searched the genome database to identify any genes or enzymes associated with the production of beta-carboline. Unfortunately, no such gene or enzyme seems to be directly related to the synthesis of beta-carboline in this bacterium. Also, my investigations regarding the McbB enzyme (which is an enzyme that works for the production of beta-carboline) have unfortunately provided no evidence of its presence in my bacterial strain.

So my question Is there an alternative methodology or approach by which I could clarify the mechanisms used by this bacterial strain to produce these compounds? For instance, the synthesis of beta-carboline usually involves the enzymatic action of tryptophan decarboxylase, which catalyzes the conversion of tryptophan to tryptamine. However, my bacterial strain seemingly lacks this specific enzyme. I am hoping that if any practical strategies or methodologies exist, I will try them as a first step to finding some answers for the synthetic pathway.

I sincerely appreciate any insights or directions you can provide.

Is taking multiple factors such as genome sequence, taxonomy levels, behavioural patterns, anatomy and etc into account when comparing 2 specimens more accurate than just comparing its genomes?

If the species is least concern according to IUCN red list, then why we go for mt-gene sequencing? What will be the usefulness for the species after research? Would it be the useful research or we should go for some other research topic?

As one of 16s rRNA sequences giving only 70% query cover. What could be the major issues with the sequencing? Suggest some laboratories in India for bacterial genome sequencing and it's price.

I am looking for a way to predict Transcription Start Site (TSS) in Aedes genome, Is there is any bioinformatics software or pipeline to predict TSS from genome sequence or a gene

Now several scRNA-seq platforms are used in Bacteria. I can not get the point.

Firstly, Bacteria have Horizontal inheritance, meaning they can exchange the genome. How to get the ref genomes in the mapping process? Is the heterogeneity due to horizontal inheritance or regulation or genome mutation？

Secondly, most scRNA-seq platforms analyze the Bacteria in the culture but not the original. What's the obstacle here？ With culture, why not use bulk seq?

Thirdly, the RNA in Bacteria may be polycistronic mRNA, how to deal with them?

Why Not Single-Bacteria genome sequence?

I need help on visualizing a microbial genome sequence obtained from Miseq-paired end reads. I am not sure, when using the tools, how the input file should look.

Can anyone help me with generating the input files? I did go through the ggbio manual, so if anyone could point out the steps to generate the files to be loaded into the respective software, that would be great.

I have been doing some qPCRs on bacterial genomic DNA. I am using 16S as my "housekeeping gene". My sample of interest, however, yield a lower CT value.

I am trying to figure out how to overcome this issue.

Should I redesign 16S primers?

Is there any other "housekeeping gene" for bacteria?

Should I send my samples to genome sequencing?

I used illumina to complete the genome sequencing of phage and the genome was assembled into one contig. How do I know if the genome I'm got is complete, and if it's linear, how do I know where it ends?

Hi!

I am actually start working with HERVs and other endogenous viruses and try to verify them on qPCR bases (depending on what part is transcribed in defined setting e.g.). Therefore i need the genomic sequences of different HERVs and other TE- elements with annotations (e.g. LTR-gag-pol-env-LTR), so I am able to design primers for different parts. Only thing i found is giri repbase, sadly our university does not have a subscription. Does someone of you has ideas, where i can get annotated consensus sequences for TE- elements and HERVs?

Thank you!

I want to make sure that my competent cells are NEB stable cells only but I couldn't find the genome sequence or NEB stable specific primers on web. Has anyone ever tried to validate their NEB stable cells via PCR? If so what is the primer sequence for it?

I have been struggling to obtain a good read N50 by sequencing genomic DNA on MinION from Oxford Nanopore Technologies with the ligation sequencing kit, and I wonder if it wouldn't be easier (quicker protocol), cheaper (no 3rd party ligation enzymes) and maybe get a higher read N50 using the Rapid kit (less washing steps and DNA loss). Can anyone share your experience? Thanks in advance.

I'm trying to design a CAR-NK cell that expresses a particular receptor. I have the entire genomic sequence of the gene for that receptor which is 10s of thousands of nucleotides long, I have the mRNA sequence, the coding DNA sequence and the amino acid sequence. Which one of these is used in a plasmid or viral vector for the expression of a construct?

I need this cell line to validate my genomic sequencing.

Genome sequence data need to be converted into numerical database to perform machine learning algorithm.

I am trying to do de novo genome sequencing for a Basidiomycetes fungal isolate using PacBio CLR, as we already failed HiFi Pacbio and still hoping to get long-read sequences. We used CTAB method together with Qiagen Genomic-tip for the HMW DNA extraction, and we saw bands > 25 kb when we run the agarose gel. After we shipped the sample to the sequencing company, they run Qubit and found the sample was degraded with the main fragment < 15 kb so it is not even worth to proceed library prep. I wonder if anyone also had the same problem with similar species. I can't figure out what caused it because we did the same extraction and shipped another isolate at the same time, that one did not have a problem and was successfully sequenced.

Hi all,

I am trying to work with SBE2 gene in a plant. I only managed to find partial DNA sequence of the gene in NCBI database. The genome sequence for the plant is available but not its genome annotation. I have a very basic background in bioinformatic and really need some pointers on how to identify the full gene sequence based on the available genome sequence. Thanks in advance guys!

I'm working on viral genome sequences (hundreds sequences) to obtain the frequency of particular mutation in that group. I'm relatively new on genome analysis field, so I have been doing it manually by submitting the sequence to a database and take record of every mutation, one sequence at a time. It takes plenty of time, and also risk of error in taking record.

Is there any software that can help me to analyze all the sequences at one time so I will be able to obtain accurate mutation frequency?

Thank you so much for responding me.

How relevant it is to carry out CRISPR/Cas based genome editing of cds obtained via De novo transcriptome sequencing? How important it is to have the genome sequence available for gene editing? What could be the possible outcomes and consequences ?

Please suggest

Regards

Alka Jangra

What are the main challenges? How are results typically interpreted?

How does this differ from genome sequence data collected evolution experiments?

hello. My laboratory is doing a Killifish genome sequencing and I don't want to lose this chance to use it and decided to start my own research. I have a research idea but I need more details for a proposal.

I wanted to know what tools are there to get proteins from DNA. And every difference between them. For example, I will not need a whole protein sequence, but specific multiple proteins (TERT, TZAP, RAP1, TRF1, 2, TPP, TIN). Is there any tool for that? To search for a specific protein.

Despite which tool I need, please tell me about any tool that is related. Thank you

For the prediction of 5s/8s, 16s/18s, and 23s/28s ribosomal RNA in full genome sequence, which server is best.

And the server whch is windows based and not Linux-based.

I have read the sentence below, and I have still diffculty to understand the term Read Depth. I would be glad if someone could explain it to me.

I would like to start a new plant phylogenomics project, but I am currently struggling with choosing the best-suited approach. My idea is to combine the latest genomic approaches (e.g., Oxford Nanopore Technology (ONT) Sequencing) with DNA extraction of herbarium specimens due to Covid-19 restrictions and thus restricted traveling possibilities.

- Do you have any experiences with DNA extractions from herbarium specimens and ONT genome sequencing?

- Assuming the availability of a reference genome, would you recommend using low-quality long ONT reads instead of high-quality short Illumina reads (also in terms of costs) from DNA herbarium specimens for reference-guided read assembly? Depending on the specimen age, DNA is already strongly fragmented and ONT won’t capture long fragments (> 1 kbp) anyway, or?…

- Are there software tools you would recommend for reference-guided ONT read assembly and generation of diverse output files for further downstream analysis (e.g., tree and structure software)?

The plant species I will use has a diploid genome size of 0.8 Gbp. The majority of samples are polyploid hybrids.

I have found recombination in a certain caliciviridae virus which was detected by sequencing its partial capsid and polymerase region. It was not possible for me to perform a complete genome sequencing at this moment. How could I do analysis with this partial sequence in SimPlot version 3.5.1? Can anyone please give me step-by-step procedure to perform the analysis? It would be a great help. Thanks.  

I want to align mRNA with the genomic sequence. I was using MGAlignit earlier but the server is not responding now. Do you know of any alternate tool?

Dear all, I am trying to use CD-hit to remove the duplicates from the file that is the output from trinity (RNA seq assembly).

I used the following parameters:

cd-hit-est -i in.fasta -o out_cdhit90.fasta -c 0.90 -n 9 -d 0 -M 0 -T 0

But the output file still contains lots of small or fragmented sequence plus the best one. How can I remove those small or fragmented duplicates by changing the parameters?

thanks

ZQ

I have tried SmartBLAST, but there I'm not getting the exact what I want. If anyone can guide me on how to get the same from ORF's will be a great help.

Also I need to find out the functional regions with high similarity and identity, so for that do I have to use MSA tool?

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I recently sent samples to be sequenced in Macrogen-Korea, but do not know much assemble secuenciasy analyze the quality of sequencing. So I want to learn.

Hi, I have the total genome sequence of an organism. I want to scan for the presence of a special gene from this sequence. How is it done, with which program should I do it? I would be very grateful if you could reply urgently.

I am going to make a list of genes with unknown functions in Salmonella typhimurium strain LT2. To make a comprehensive list, I wondered if someone knew a publication or database that could help me.

I have the whole genome sequences (WGS) of 200 strains of the same species, and I want to compare the protein sequences of a specific gene among 200 strains. I would like to ask is there any software that can automatically extract the protein sequence of the target gene from 200 WGS simultaneously, or I need to manually find and extract the protein sequence of each strain and then do the protein alignment? Thank you in advance.

For Bio-synthetic gene cluster analysis, many tools required protein input sequence that's why we have to submit protein sequence for analysis of putative gene clusters.

Hi all,

I would like to now if you have any information related to this issues, more precisely companies who could provide services for

1. genome sequencing and assembly ;

2. whole methylation sequencing  for 20 samples including bioinformatics analysis

Thanks

I found that in NCBI, sometimes it is hard to find the complete genomic sequence of a gene, does anyone know if there are other websites good for searching for genomic sequence? Thank you.

I have the Illumina whole-genome sequences data (which included both chromosome and plasmids sequences) of over 100 strains of the same species. Could anyone please tell me how to make a phylogenetic tree based on only core-genome sequences (only include chromosome sequence)?

I have 182 whole-genome sequences including 181 reference genomes and a genome sequenced us, I would like to draw a phylogenetic tree using these 182 whole-genome .fasta files. I tried with MEGA X but not running even using a server. Can anyone suggest a programme or online tool for this?

I am having an assembled fasta file (~3.7 GB in size). I tried using the web based MISA v2.1, but it accepts fasta upto 2Mb only. Is there any other alternate software which can be used for a large size dataset for filtering SSRs?

I am writing a research article using complete mitochondrial genome sequences. I need to reconstruct the phylogenetic tree using the MrBayes.

Hello everyone!

I want to generate a phylogenetic tree of my 16S rRNA sequence but NCBI shows similarity with compelete genome. When click on accession number it goes to complete genome page. Can anyone suggest me some methods to do this! The sequence is attached.

Thanks

I am working with house cricket sequences and already search SSR for population genetic studies.

Population study is taking time so i wanna make use of genome sequence for publication.

Regards

Yash M Gupta

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.

I have whole genome data sets of SARS-Cov2 of different countries. Now I want to exclude genome sequences which represents 100% sequence similarity. I want to do it by clustering analysis..

1) Can anyone suggest me any tool that is good for Viral whole genome clustering analysis??

2) any another method, that i should know??

A big thank you in advance.

I am preparing a research article on the complete mitochondrial genome sequences of Noctuid moths. How to find the tandem repeats in the A+T-rich region. Kindly guide me.

I have RNA-sequence/est and genome sequence. I would like to identify the intron splice site with 5' GT-3'AG bias

I'm trying to identify certain amino acid sequences that occur in the ORFs generated from an annotated genomic sequence. For instance, I would like to find any occurrence of LPXTG (where X is any amino acid). Can you recommend a website or software that will allow me to do this? Thanks.

I want to know about the current status of plant genome sequencing in this world. How is the reference genome in this case? Does anybody have any knowledge regarding the topic of Plant genome sequence?

In the aftermath of the 1918 Spanish Flu pandemic there was a marked increase in incidence in psychological and psychiatric illness incidence. These conditions now often referred to generically as post-viral syndrome increased hospital admissions and treatment of mental health disorders in the years following the outbreak in 1918.

Population studies in countries that did not take part in World War One seem to indicate that the possible post war melancholy could be ruled out as a confounder as this increased incidence was seen in all countries affected by the flu that had been non-combattants in WW1.

Could there be a lasting and chronic element to all SARS type respiratory disorders?

SARS genome sequences have been detected in the brain of earlier SARS autopsies with LM, EM, and with real-time RT-PCR. The signals were confined to the cytoplasm of numerous neurons in the hypothalamus and cortex. Oedema and scattered red degeneration of the neurons was identified in the brains of 80% of the confirmed cases of SARS examined.

SARS viral sequences and pathologic changes have not been found in the brains of unconfirmed cases or control cases.

We may have a longer lasting health care problem that will affect those 'recovered' from Covid-19 for some years to come.

Hi!

I'm looking to use Oxford Nanopore technology for a project I'm working on- I'll need to sequence phage genomes (from faeces) - both RNA and DNA. Our lab already has the MinION Mk1b application but I've not used it before. If anyone has done similar work i'd be really grateful for any general advise or warnings of pitfalls to avoid. Very open question - but would be helpful to learn from experiences!

Thanks Aoife

I want to run a variant caller on ASD sequences but I can't find any database that allows open access to it. Have we got something like SRA to download such data?

I want to download chromosome wheat genome sequence v 4 from IWGSC website but can't find a way to do so. Secondly I want to perform BLAST search using GENEIOUS software can some1 suggest any other appropriate software to BLAST, in which I can compare genetic architecture of different Spp. (rice, maize, wheat, barley) ?

I have identify genome sequence of my bacterial through 16S rDNA sequence method. since my bacterial is a hydrogen producing bacterial, would there be a software that i will insert those sequences and it will show up the presence and position of hydrogen producing proteins from the sequence

I have a genome sequence, I wish to draw a phylogenetic tree to compare it with similar genomes, How can I do that?

We have whole genome sequence data of Paenibacillus sp. and Arthrobacter sp. On submission of the sequence data with NCBI we got queries (a) If this is a circular genome, are the ends of the sequence contiguous around the circle with no overlap and no gap? (b) Should there be a gap at the end of the sequence? If there should be a gap, do you know the gap size or should this be an unknown length gap? 

I would like to get quotations for whole genome sequencing (WGS) of human Required depth of coverage for WGS: 30X and for WES: 200X Sample source: Blood

I am trying to use BLAST tool to screen my own genome sequences for specific genes.

I am lacking bioinformatics knowledge and would like a simple explantation on how to do this.

Briefly, I've been given the genomes of the strains I am working with and would like to check whether they carry some known genes (NCBI) for specific compounds.

Any clue on how to do this?

Dear all,

I have the following troubles:

I am trying to go from cDNA sequence to genomic in order to locate some mutations. I have the reference sequence

and the corresponding genomic sequence:

I am trying to locate several mutations, but if anyone can show me how to get to one or two then I will do the rest myself. The mutations I am looking for, in this instance, are c.192+5T>TA (this is an intron variant) and c.805A>T. I guess the TA refers to a heterozygous sample. Although I can (probably) find the c.192 and c.805, I cannot locate the mutated nucleotides at all on the genomic sequence. So the question is where do I get it wrong, either in looking at the coding sequence in the wrong way or not looking at the introns in the correct way? Can anyone help me on that? Much appreciated.

Hi all,

I am interested to do a comparative genome analysis using EDGAR comparitive genomic tool. I have a novel bacteria whose genome sequence was obtained by us and now I was to do compare this novel strain´s genome to it´s phylogenetically closely related neighjbours. This bacterium belongs to the genus of Paracoccus.

I notice that EDGAR allowed to select the genomes which are already provided in their database but does not allow to add a sequence of any genome (may be I missed?). Could you please suggest me how can I do a genome comparison using thsi tool or if you can suggest some other tools (in case EDGAR does not allow to add new WGS to their database for comparison)?

I know about BRIG but that is not as cool as EDGAR since it provides Venn diagrams and many other results for better interpretation.

Thank you and Regards,

Timsy

The rationale would be to have enough confidence in calling a variant. Thanks, all!

I need a dataset with known organism information (which organisms are in the dataset, it's best if the coverage of each organism is known). I did a search on the web but did not find any.

The results show me the presence of plasmids, and compares them with plasmids from the database, but I can't find their locations, thank you!

Is there any way to predict telomere sequences from genome sequencing data?

Considering the use of this kit ( SQK-RBK004 ) for cost efficient sequencing of multiple bacterial genomes per flow cell. Any comments on its efficacy etc. would be welcome! Thanks!

I am preparing a research article about the complete mitogenome of moth. I want to include the details in my research article.