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Questions tagged [pcr]

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1 vote
0 answers
28 views

How to shade text in a table

I have currently made a tool that takes input sequences and maps them to a reference sequence highlighting it. You can then save the word document and it creates a table of the sequence name and the ...
1 vote
1 answer
31 views

PCR primers for detecting hetero vs homozygous and WT mice in our colony

My PI has asked me for advice on making a PCR screen to identify mice from a particular Cre line. We have multiple Cre lines in the colony so it can't just be for mice with a copy of Cre in their ...
2 votes
1 answer
162 views

1.What size PCR product will be generated using the primers (bold and underlined) in the sequence below?

I would like to know the steps of this question and how to determine the forward and the reverse primers and also the length of the sequence, without using NCBI. If you could explain and give an ...
1 vote
1 answer
89 views

Do we design primers for single stranded DNA with same criteria as we do for double stranded DNA?

I am trying to design primers for a single stranded DNA virus. But I am having a dilemma as whether we design the primers same way as double stranded DNA? do we have to design forward and reverse ...
1 vote
1 answer
70 views

Choosing the best number of species for assay

I am doing amplicon sequencing of a virus across many different regions. Lets say I have 20k unique variants of the same virus that I put into my pcr assay and after sequencing and amplifications I am ...
3 votes
1 answer
357 views

Tool to remove a PCR contamination in NGS data

BACKGROUND I work on NGS data (illumina paired ends reads) coming from a full extract of RNA (metagenomic). We are interested in the viral fraction of this extract. I observed a contamination with a ...
5 votes
1 answer
72 views

Correcting for noise in RT-qPCR gene expression data

I have a training set of RT-qPCR gene expression data (not run in triplicate) for a batch of samples with two phenotypes $A$ and $B$ on which I've trained a "logistic regression classifier". ...
3 votes
1 answer
55 views

How to optimize the number of amplicons ordered for a PCR wet experiment with several genomic ROIs?

Imagine we have an experiment for which we would like to minimize the number of purchased primers. Let's look at the layout of a single primer below: Assume we have a precomputed set of all necessary ...
1 vote
1 answer
286 views

Need a alternative or more complex version of venn diagram in python for matching dna sequences

I am in google colab and I have combined and set up a data frame list of sequences that goes like this Location ID Sequence 1.1 ........ A ........ AAGAGATA 1.2 ........ A........... ...
1 vote
3 answers
146 views

K means clustering, would PCA be a better option?

I have the data below. I need to use a clustering method to classify them and into categories of "Heterozygotote, Allele 1, Allele 2 and No Call. The values in RFU1 and RFU2 are used to determine the ...
1 vote
1 answer
60 views

In silico PCR: any models that can quantitatively predict the initial content from the end amplified result?

Let's say I've run the PCR and I now have a certain amount of the amplicon product. Is there any model that infers how much of it was there before the PCR started? I suspect it should be a ...
5 votes
1 answer
4k views

Why do NEBNext indexing primers have sequence between the p5 oligo and index?

In a previous post I asked Why do NEB adapters have non-complementary sequence? Since then, I realized that there is some other sequence in the p5 indexing primer, as well as in the p7 indexing ...
1 vote
1 answer
179 views

How is PCR duplication rate computed in scATAC-seq?

Reading Cusanovich et al. (2015) I encountered the sentence: We mixed pairs of cell lines (HEK293T or HL-60 versus GM12878), performed combinatorial cellular indexing, and sequenced the resulting ...
1 vote
2 answers
3k views

Why do NEB adapters have non-complementary sequence?

I am making some infographics of different library prep types and noticed something weird about NEB's Ultra II adapters. Molecule 1 is the desired product of the ...