Questions tagged [dna-sequencing]

Technique(s) by which the sequence of DNA is obtained. The principles are similar for [tag:rna-sequencing].

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Why are a many copies ending at _each_ location needed (Sanger method), instead of just regular PCR?

Apologies since this is probably a very basic question : The Sanger sequencing method creates large numbers of sequences of all possible lengths, ending with a specific nucleotide, by terminating ...
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How to convert enrichment/depletion to frequency for comparing deep sequencing to sequence profile?

I have two datasets, from different sources, that I need to compare. The first set is deep sequencing results of a directed evolution experiment, where I have the naive library and selected library ...
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Can 1.5 gigabytes encoded in the human genome really account for the complexity of a human being?

I read the human genome is 1.5 gigabytes in size. Thats actually not a lot; Photoshop probably takes more space. Mac OS takes 10+ gigabytes of space. Also, the genome is 1.5 gigabytes when counting ...
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What does the final (Illumina) RNA-seq library contain? ss cdNA or ds cDNA?

I guess the oligos on Illumina's flow cell only hybridize to to single-stranded cDNAs (sscDNA). However, when I read the TruSeq protocol (googled: TruSeq® RNASample Preparation v2 Guide) at the PCR ...
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How to get phased haplotype from vcf file?

Although I'm reading all days and nights, still confused in this area. I have hundreds of BAM files (from whole-genome sequencing of a given human population). I was wondering how I can obtain phased ...
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Single-cell ATAC seq arrays

As part of a data analysis project, I encountered two kinds of single-cell assays for transposase-accessible chromatin using sequencing (single-cell ATAC seq) methods. The first uses combinatorial ...
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1answer
86 views

What does it mean to combine in-solution capture of chosen RAD tags to target sequencing reads to desired loci?

The article about "rapture sequencing" found here says: "This protocol also recovers more unique (nonclonal) RAD fragments, which improves both standard RAD and Rapture analysis. Rapture then uses ...
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In a Chromosome, 2 nm is the length of what?

this figure comes from the nature 2 nm at the top right hand corner is the length of what?
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How DNA probe binds

I am studying about southern hybridization now and I've a doubt.After the DNA has been fragmented using restriction enzymes and obtained on nitrocellulose blot , it is still double stranded (the ...
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Why are there two abrupt changes in the genome sequencing price curve?

In the price curve of the genome, from NIH: NIH explained the abrupt drop at 2008 thusly: beginning in January 2008... the sequencing centers transitioned from Sanger-based (dideoxy chain ...
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What percentage of the human genome hasn't been sequenced yet? If percentage estimates aren't precise, why is it difficult to estimate?

What percentage of the human genome hasn't been sequenced yet? I have read different estimates, e.g.: https://www.genome.gov/human-genome-project/Completion-FAQ (mirror): "In the April 2003 version, ...
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158 views

What is a sequencing artifact?

I'm rather new to bioinformatics, so this might be a rather basic question, but what are sequencing artifacts, in the context of variant calling?
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Why does one combine PCR and cloning as ways for amplification of sequences?

Why does one combine PCR and cloning as ways for amplification of sequences? Don't they produce the same result? I was reading the paper https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1864885/ and got ...
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If some humans inherited 3% of Neanderthal DNA, why are we 99.9% same genome? [duplicate]

Many sources say that humans are 99.5 to 99.9 percent the same. Also some sources state that some humans have 3.4% Neanderthal DNA and some don't share those genes. Why is that?
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Are SNPs and alleles the same thing?

It seems to be quite difficult to find an answer to this. Are SNPs the same thing as alleles?
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Why are two forward and reverse priming sites depicted here? what do they do?

This question is in regard to the baculovirus expression system. (source: amsbio.com) Why do we need to generate primers for the polyhedrin promoter and the baculovirus? Primers are needed to ...
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What are primary reasons for the failure to localise/anchor sequences in genome assemblies?

My question concerns the incorporation of individual sequence reads into chromosomes during gene sequencing projects, especially those with larger genomes such as Drosophila melanogaster or Homo ...
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Does increasing the value of k in k-fold coverage requires more memory?

Study: Here is what is k-fold coverage which is know as sequence coverage. According to this, 2-fold coverage means the whole DNA is sequenced more twice in average. Question: If I have a genome ...
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What's the difference between shotgun sequencing and clone based sequencing?

In a lecture during my undergraduate degree we were introduced to the race to complete the human genome. Celera were competing with Sanger and collaborators to sequence the human genome. Celera ...
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Are the Fastq files content is truly random

I'm building a system to generate fastq files as they are being output from HiSeq 3000 and HiSeq 4000. This will serve us to test our internal systems with some gold standards. As we are working with ...
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What happens after the purification step in Hi-C sequencing?

I am a statistician reading an article on Hi-C, and I am trying to better understand one of the steps in the DNA isolation and sequencing process. Since I'm a statistician, please try to avoid too ...
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What are haplotype blocks and what is the effect of hybridization on these?

In this PDF, there is a quick definition of haplotype blocks. A haplotype block is a set of closely linked alleles/markers on a chromosome that, over evolutionary time, tend to be inherited ...
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If we sequenced the genome of every species, would all phylogenies agree?

The Tree of Life is still up for debate. Most of this debate seems to be due to a lack of genomic information, but that deficiency is decreasing rapidly with advances in technology and sequencing ...
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Construct picture of person's face from DNA

Would it be possible to construct a picture of a person's face from his/her DNA?
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How much of my ancestry will match with my brother?

Recently, my brother (full sibling) got his ancestry checked from MyHeritageDNA. They have a similar service like 23 and me and I've found out that both companies are offering the basic service almost ...
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Why do genetic testing companies (FTDNA,AncestryDNA,23andme) express DNA shared in centimorgans (cM) instead of in number of base pairs or in percent?

Expressing DNA shared in number of base pairs or in percent of total genome would be so much simpler and easier to understand for the customers. .
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How many palindromic sequences in human genome?

I'm writing an article on palindromes (the words) and I wanted to mention the existence of palindromic gene sequences. Roughly how many palindromes exist in the human genome? I understand the number ...
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A cure for radiation exposure?

Some of the greatest inventions of my lifetime have been from looking at nature and copying. I found out tadigrades in outer space have the ability to repair their own DNA when exposed to radiation ...
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Do we posses technology to rebuild sequenced DNA? [closed]

Is there existing technology that would allow to rebuild DNA molecule from a digital file storing sequenced DNA? If not, does there at least exist theory explaining how such device could be built?
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Why is GenBank growth slowing down?

https://www.ncbi.nlm.nih.gov/genbank/statistics/ shows the growth of the GenBank database is slowing since WGS (Whole Genome Shotgun) emerged. Is this happening because sequencing centers are ...
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Parameters of Variant calling analysis [closed]

What is the good or stringent parameter for variant calling? At present using the DP > 10 and Q > 30 for Variant calling. Is it ok?
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What is mRNA expression level?

I cannot find a clear explanation of what is mRNA expression level, and how to measure it. I would appreciate if someone explained it or gave a reference.
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What is the structure of DNA during Interphase? Chromatin Fibre or Beads on a string(nucleosomes)?

During Interphase, does DNA predominantly exist as Chromatin Fibre (30nm fibre) or Chromatin/beads on a string structure(10nm) ?
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Must I HPLC-purify my PCR Primers for amplicon sequencing with PacBio SMRT?

I would like to order some primers for amplicon sequencing. I am using universal tag primers (unusually long 30-mers recommended by PacBio for SMRT, both F&R) + barcodes (16-mers). The resulting ...
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2answers
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Meaning of Phred score - probability of base calling error

I learned that Phred quality scores are logarithmically linked to error probabilities. For example, if Phred assigns a quality score of 10 to a base, the chances that this base is called incorrectly ...
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How does Sanger sequencing resolve two bands with equal size? [closed]

In Sanger sequencing, how can we read the sequence if we have 2 bands with the same size?
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Sequence of ribosomal RNA

Is it possible to sequence rRNA directly, that is, using the ribosome rather than the DNA from the nucleus? For example, this paper, Complete nucleotide sequence of a 16s rRNA gene from E. coli, ...
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What causes skewed lanes in a DNA gel electrophoresis experiment?

In gel electrophoresis, what causes effects like these (see column 11 in the first one, and column 6 in the second)? (These images were samples that I took from an online activity we did for class)...
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Translocation and Isolation of Specific Bacterial Codons

I am currently working on a research project in which I am aiming to translocate a gene from a specific bacteria (strain NT-26, an arsenic oxidizing bacterium) into another bacterium. I am doing this ...
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1answer
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How does a polymerized TruSeq adapter affect sequencing reads?

I am trying to build a NGS library on enriched DNA (DNA length = 93 bp). I am using PCR amplification with two overhanging primers, both containing Illumina adapter sequences (Universal Adapter & ...
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Is it possible to deduce facts about a person's parents just by studying his/her genome?

As an example, suppose Anne had abusive parents. Is it theoretically possible to deduce this from her genome even if she didn't inherit this quality (of being an abusive parent)?
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denovo 2Gb whole genome sequencing on a small sequencer

What are the limitations of using Illumina's smaller sequencers (e.g., iSeq100, miniSeq) to do de novo whole genome sequencing on an organism with a larger genome (~2Gb)? Naively, it seems even if I ...
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What experiment for first time showed DNA is anti-parallel? [closed]

Scientists how to prove DNA is anti-parallel?
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Any examples of a group of micro-organism that continuously spans two or more species classifications?

Are there any examples of a group of micro-organisms where two different, established species are designated and these two groups meet all aspects of the definition of species (perhaps can not ...
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What is the strand specificity of a reference genome?

It's a simple question but I've come across many people who have this question, is the reference genome Positive of Negative strand? Indeed, I've had heated arguments over the same issue. So here's ...
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DNA preservation at room temperature

I am considering preserving the DNA of a family member who passed away. The funeral home offers a service called DNA Memories from a Canadian company (CG Labs). They have two options: Store the DNA in ...
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Sequencing inaccurate at the primer site

The times I have sent a sample to sequence, both the forward and the reverse primer sites, show high inaccuracy while the rest of the gene is correctly sequenced. Because of this, the sequences of my ...
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Error frequency of DNA replication without proof reading activity of DNA polymerase?

Different domains of DNA polymerase contain different activity, like 5'->3' polymerisation and 3'->5' proof reading ...
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2answers
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Pyura DNA extraction

I'm am struggling with genomic DNA extraction from different samples of Pyura chilensis; the DNA is degraded as can be seen on the gel. We've always used GeneJET Genomic DNA purification Kit (by ...
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1answer
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Is it possible to re-align of SAM/BAM files?

I am carrying out some metagenomic comparison using whole genome sequencing approach. I would like to filter out reads from my files in a sequential manner, thus I would like to align to a first ...