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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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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 ...
14
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2answers
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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 ...
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 ...
9
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1answer
502 views

Why does this human bam file only have one copy of each chromosome?

As we know that in human DNA sequence, one copy of chromosome comes from mother's DNA and another copy comes from father's DNA so as to form two copies of each chromosome in human DNA. So, if we ...
9
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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
votes
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 ...
8
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3answers
1k views

What is the index fastq file (sample_I*.fastq.gz) generated when demultiplexing Illumina paired-end runs?

What is the index fastq file that comes with some Illumina sequencing datasets? (The samplename_I*.fastq.gz file.) For example, I recently received some 10X ...
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) ...
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? ...
7
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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 ...
7
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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....
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 ...
6
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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 ...
5
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6answers
282 views

Identifying Indels from Chromatograms

I have around 100 chromatograms (.ab1 files) from Sanger sequencing a genome at loci believed to have an indel. I'm new to interpreting this kind of data in ...
5
votes
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, ...
5
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2answers
87 views

Deciding which samples go in which batch

I have 370 samples to sequence, we probably will end up using only 96 samples per run (due to the barcode with the primers we'll use). This means running 4 batches. To minimize the batch effect I need ...
5
votes
1answer
192 views

Can a data file in VCF format be converted into FASTA?

I'm considering purchasing the 'MyGenome' product by Veritas Genetics to analyze my genome for a project. I'd like the data to be in FASTA format, but Veritas only provides VCF data. Is it possible to ...
5
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1answer
83 views

Extracting sequences from FASTA beginning with common 5' end

I am trying to figure out the best way to extract sequences from a FASTA file which begin with a common 5' region of 43 nucleotides. Preferably, I would like to to allow for "fuzziness" in this region ...
4
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1answer
333 views

Why do I get so many insertions from Minimap2 on my Nanopore WGS?

I'm a starting my analysis on nanopore whole genome sequencing. I start my analysis from this popular Github. The sample I downloaded was WGS for NA12878, so I would assume it's alignment to GRCh38 ...
4
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2answers
85 views

Use of heterozygous SNPs in cancer research: why?

When reading about allelic fraction (AF) and SNPs in cancer research, they always mention the fact that they're using heterozygous SNPs (informative SNPs). Why is this? Why can't we use homozygous ...
4
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2answers
747 views

Can I stop my nanopore sequencing run if there are no more reads being produced?

I am sequencing a whole genome on MinION. I have used this flow cell for 6 hours for the phage lambda control. That gave me around 300,000 reads. I started a 48h sequencing run on the same flow cell ...
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, ...
4
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1answer
78 views

Retrieval of genomic position by using biomaRt package

I have a list of several protein names, their primary gene name and their amino acid sequence (as extracted from the 'SEQRES' section in their corresponding PDB files). I'm looking for the genomic ...
4
votes
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 ...
4
votes
1answer
60 views

Are there any Genome in a Bottle-like resources for non-humans (especially for invertebrates)?

Genome in a Bottle is an excellent resource that provides many types of DNA and RNA sequencing reads for a single individual/cell line to test genome assembly and analysis tools. For example there are ...
4
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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, ...
3
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3answers
96 views

Tools for quality trimming at 5 prime?

I'm looking for a tool that is capable to do quality trimming at 5' end and it is configurable. For example, I can choose the quality trheshold, the read length, etc. Any recommendations? I'm ...
3
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1answer
376 views

Significance and timing of “mux scans”

I'm using MinIONQC to do quality control on some ONT data. The software plots several read characteristics over time (hours passed during the sequencing process). These plots contain several vertical ...
3
votes
2answers
100 views

Separation of mixed plasmid DNA sequences post whole-plasmid sequencing

Imagine a DNA sample containing a mixture of different intact plasmids. These samples are sequenced using either MiSeq or HiSeq sequencing. Would it possible to assemble these plasmids post-sequencing ...
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 ...
3
votes
1answer
108 views

Paired end sequencing with R1 and R2 of different length. Possible?

There are commercial sequencing kits/sequencers that allow for paired end sequencing in which the two reads obtained for each fragment are of different length? For example 150bp for R1 and 50bp for ...
3
votes
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: ...
3
votes
1answer
58 views

Produce a single sequential FASTA sequence out of BAM

I'm having problems properly looking for a solution because I'm a layman in Bioinformatics not familiar with the terminology. I'm hoping you can nudge me in the right direction, please! Thank you very ...
3
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1answer
55 views

Mixcr batch processing

I have a folder with 150 pairs of fastq files from an illumina sequence run. I need to precess them with mixcr, how can I do this in a batch with a single command?
3
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1answer
214 views

How many reads do I need to cover the entire genome?

Suppose my genome is 3 million bases and that my reads are 100 nucleotide long. I need to know how many reads I need to cover the entire genome. I start from using the equation $C = \frac{N \cdot L}{...
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 ...
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 ...
3
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1answer
73 views

Difference between copy number neutral reads and active reads

In this paper, the authors talk about copy number neutral reads (as reads that equally represent parental chromosomes) and active reads (as reads from only one parent chromosome): We reasoned that ...
3
votes
1answer
108 views

How to create a nexus file for BEAST?

I am learning to use BEAUTI and BEAST software. I followed their tutorial and was able to run example data without any problem. Now, I want to do phylodynamic analysis on my data. I have downloaded 70 ...
3
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0answers
18 views

dog coordinates (canFam3) to human coordinates (hg19)

I've converted dog coordinates to human using UCSC LiftOver. These are 200bp intergenic regions that are differentially methylated from normal dogs to cancer dogs. I've converted these to human ...
2
votes
2answers
57 views

How to best detect the “peaks” in RNA-seq data that are not assigned to any gene?

I encountered that many reads from single-cell RNA seq data were lost in the analysis because not assigned to any gene (genome: galgal6). I am trying to find an approach than could give me all the "...
2
votes
2answers
360 views

Hardware Requirements (specs) for Bioinformatics-dedicated desktop [closed]

I know this is a somewhat general or vague question, but I’m interested in your opinions. I must build a desktop for general bioinformatics activities with human genomes. I will work with Python and R ...
2
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1answer
511 views

Why are there more barcodes than GEMs in 10X chromium data?

Question: Why are there more barcodes than GEMs in 10X chromium data? Introduction I have several 10X genomics chromium libraries made with the de novo assembly/genome protocol. The 10X chromium ...
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 ...
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 ...
2
votes
1answer
84 views

E.coli Sequencing & Analysis

I have been given the task of assembling a 'new' Ecoli genome and analysing the genes present etc. The Ecoli is a new strain, and has been taken and run on a Nextseq 500 in high-output mode with ...
2
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0answers
45 views

High percentage of poly A sequences in 10X chromium R2 read

I'm currently analyzing two samples of eosinophil cells isolated from mouse lung and the samples are of very different quality. According to the Cell Ranger summary 56% of the reads can be mapped to ...
1
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2answers
1k views

What is the name of this type of figure?

This figure comes from this wiki page. I googled "gene location figure" and "gene location plot", none gets similar results. I also tried search by image, none of the results is close. so, what is ...
1
vote
3answers
160 views

Is there a read mapper that allows input in SAM format instead of FASTA/FASTQ?

When we get raw sequenced reads in FASTQ format, we usually need to do some pre-processing (QC, demultiplexing, etc.) prior to sequence alignment. The only way to register the results of this ...
1
vote
1answer
78 views

Why do ten rows (Figure_1) correspond to 2 bits (Figure_2) in a sequence logo?

Following this question, I'm confused with the computation of sequence logo Following data comes from the book "Machine Learning - A Probabilistic Perspective (Figure_1)" here is the corresponding ...