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Questions tagged [sequencing]

DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule.

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3
votes
1answer
697 views

How many reads has my sequencing run produced on minion?

I am running a 48 h sequencing protocol on a FLO-MIN107, on MinION, with DNA library-prepped with SQK-RAD004. MinKNOW is installed on my Mac and controlling the MinION. I have found the following ...
4
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2answers
541 views

What does it mean to over sample?

One of my studies (A) involve sequencing the microbiome. After selecting the variable region and the primers for the targeted region of the 16S sequence. The samples were sent to a platform. For ...
1
vote
1answer
2k views

Set directory where MinKNOW writes FAST5 files

I would like to set the folder in which MinKNOW writes the raw data. How can I do this? I do not currently know where MinKNOW will output my data. What is the default directory where MinKNOW outputs ...
5
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2answers
196 views

Can I use my computer while MinION is sequencing without negatively affecting the run?

I have a Mac Book Pro and I am about to start a sequencing run on the MinION. MinKNOW is going to be running for 2 days. Can I use my computer during sequencing? Can I browse the web and use Excel, ...
0
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0answers
32 views

I have two files: mys.nuc and ms.prep and need to use bioseq to extract the ORF sequences and save as “mys.nuc2”

Below are the two files I am working with: ...
2
votes
1answer
236 views

What to do with the configuration test cell after configuring the MinION with it?

What to do with the configuration test cell after configuring the MinION with it? I received a MinION with a configuration test cell in it. After running a configuration in the MinKNOW software while ...
7
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4answers
4k views

What are the pros and cons of the different basecallers in Oxford Nanopore Technology Sequencing?

What are the pros and cons of the different basecallers in Oxford Nanopore Technology Sequencing? I am about to start a MinION run on my laptop. What should I consider when choosing my basecaller? ...
-2
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2answers
171 views

Web scraping in R

I have ~130 gene names of different human proteins. I'm looking for a convenient and systematic way to locate each gene's location in the genome, extract its CDS sequence and find whether there are ...
2
votes
1answer
60 views

COSMIC Genotypes and Phenotypes

I'm trying to find files containing genotypes and phenotypes for cell lines in COSMIC. On their downloads page I found links for mutation data, which I guess could be converted to genotypes, but I ...
0
votes
1answer
86 views

What is the --rev_comp_mapping_barcode parameter in the QIIME1 script extract_barcodes.py?

I'm using the QIIME1 scripts extract_barcodes.py to extract barcodes from a dual-indexed MiSeq library, and then demultiplex my reads using split_libraries_fastq.py. I have been having trouble ...
0
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1answer
32 views

Identification of Microorganism

I have the 16S DNA Sequence of a Microorganism. Can I confirm its identity by only running a BLAST or do I have to follow other methods. If so please mention the methods.
3
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3answers
286 views

sort a fasta file containing the Oxford Nanopore Technologies (ONT) header by sequencing start_time ascending

I have basecalled ONT reads and converted them to multifasta. The multifasta contains the original ONT headers in this format: ...
0
votes
1answer
86 views

Coordinates to Genome R Studio Script?

I have a very, very long list of genome coordinates of areas. I need to gather a sequence in order to compare it to another database of sequences that I have. How do I produce the genome sequences ...
1
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2answers
63 views

What are Approximate Read Counts (Library Sizes) and Lengths (Insert Sizes) for Next-Generation DNA Sequencers?

I am performing a simulation study and am curious about the parameters of my simulated metagenome. What are the library and insert sizes of some of the most "used" sequencers. Mainly, I am ...
7
votes
2answers
2k views

How do PCR duplicates arise and why is it important to remove them for NGS analysis?

I am trying to understand PCR duplicates in NGS analyses (actually whole-genome). I searched, and the best answer I found is in this blog. However I don't understand if I understood how PCR ...
6
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3answers
662 views

What is deep sequencing?

People talk about deep sequencing. Is there any way to calculate how deep the sequencing is ? What should be the optimum depth to get reliable data ? I am doing whole genome sequencing of a virus ...
0
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1answer
55 views

Why do reverse radtags have different start points in radtag sequencing?

Why is it that when doing paired end Radseq that R1 and R2 have different start points? For instance, while doing Radseq you do a restriction digestion of your DNA using enzyme X- shouldn't the ...
3
votes
1answer
188 views

genes with highest RPKM/FPKM for a human RNA-seq experiment?

Which genes/transcripts show up as having the highest RPKM/FPKM on a human RNA-seq experiment? Is it usually the same genes/transcripts? EDIT: mRNAs (e.g. poly-A RNA-seq preps) and not including ...
6
votes
1answer
143 views

checks for spike-in sequence controls

I would like to know what do people verify when designing/using spike-in controls, to be used in sequencing experiments (mainly Illumina). So far I came up with this list: Does it align only to a ...
4
votes
1answer
294 views

How to confirm exon shuffling in a gene?

Note: this question has also been asked on reddit I'm trying to confirm that the sequence of a novel gene is derived by exon shuffling between several different genes. I have the promoter sequence, ...
24
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4answers
4k views

Why sequence the human genome at 30x coverage?

A bit of a historical question on a number, 30 times coverage, that's become so familiar in the field: why do we sequence the human genome at 30x coverage? My question has two parts: Who came up ...
9
votes
2answers
1k views

How do you generate read-length vs read-quality plot for long-read sequencing data (e.g., MinION)?

How do you generate read-length vs read-quality plot (heat map with histograms in the margin) for long-read sequencing data from the Oxford Nanopore Technologies (ONT) MinION? The MinKNOW software ...
9
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1answer
1k views

Which quality score encoding does PacBio use?

Do you know which quality score encoding PacBio uses now? I know some of their file formats have changed in the past year or two, but I haven't found much on their quality score encoding. The most ...
0
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2answers
96 views

How can I find the relevant pathway map from gene-gene or protein-protein interaction list? [closed]

I have some difficulties to understand/interpret the pathway map and how a gene-gene interaction list or DNA sequencing can map into pathways. In addition what's the difference between MARK/ERK ...
7
votes
1answer
135 views

What is a good pipeline for using public domain exomes as controls?

I'm currently attempting association analysis with an extremely small set of patient exomes (n=10), with no control or parental exomes available. Downloading the ExAC VCF of variant sites (http://exac....
14
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2answers
2k views

How can we distinguish between true zero and dropout-zero counts in single-cell RNA-seq?

In single-cell RNA-seq data we have an inflated number of 0 (or near-zero) counts due to low mRNA capture rate and other inefficiencies. How can we decide which genes are 0 due to gene dropout (lack ...
4
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2answers
354 views

Why are there missing calls in a VCF file from exome sequencing?

My data is a VCF file generated from an exome sequencing variant call pipeline. I'm not very familiar with the sequencing and variant calling process. I noticed that there are some missing genotypes, ...
3
votes
1answer
42 views

How do I validate a single sample ArrayCGH result?

We have arrayCGH (aCGH) results for one sample. There is a 0.5 Mb terminal duplication on chromosome 19 (62995490-63407936, according to NCBI36/hg18). The duplication is rare: a literature review ...
8
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2answers
216 views

How to transfer gff annotations in genome with extensive duplications?

Microbial genomes can contain extensive duplications. Often we'd like to transfer annotations from an annotated species to one that is newly sequenced. Existing tools (e.g. RATT, LiftOver, Kraken) ...
12
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2answers
2k views

Is it possible to perform MinION sequencing offline?

I vaguely remember, that the original plan of Oxford Nanopore was to provide cheap sequencers (MinION), but charge for base-calling. For that reason the base-calling was performed in the cloud, and ...