Questions tagged [sequencing]

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

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41 views

Genotyping technologies do not maintain phase?

Why exactly does genotyping not maintain phase? I guess I'm more confused on what's the difference between sequencing and genotyping, and as a result how phase is maintained in one, but not the other. ...
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Short reads vs long reads with regards to break points

My professor in class said the following statements. Short reads can map break points Long reads can disambiguate repeat related issues I'm very new to genomics, so I don't quite understand the ...
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133 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 ...
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1answer
657 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 ...
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1answer
62 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 ...
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2answers
123 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 ...
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1answer
133 views

Where to Download Cancer Raw Reads (fastq)?

Does anyone know where I can download cancer raw reads (fastq files), tumor and Germline for non-humans? I wanted to make a study with human data but I don't have the control access to download raw ...
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199 views

How to cluster the human genes by pathways/system-biology/metabolic properties?

I would like to make a chord plot from my data. I have a list of genes that according to my experiments are divided into 64 clusters of enrichment pattern. I would combine my 64 clusters with a ...
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1answer
493 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 ...
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2answers
2k 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 ...
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59 views

How to score how densely bases are distributed along a given oligo?

Suppose I have a short chain string of oligos, and I want to rate them from 0 to 1 based on how clustered the "A"s are in respect to the chain. For example: ...
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1answer
580 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 ...
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607 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 ...
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Sequencing Deciduous Plants

Firstly, I'm personally not in the field of Bioinformatics, so apologies if this is a dumb question. Does anyone know of any research that has been carried out on the sequencing of deciduous plant ...
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4answers
224 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 ...
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1answer
126 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 ...
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1answer
478 views

What i5 index should I use on the Illumina sample sheet for an unindexed p5 primer?

I have an upcoming run on a HiSeq X and most of the libraries in the pool have both i5 and i7 indices. However, some of the libraries were made with the IS4 p5 oligo and it is unindexed. The IS4 ...
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1answer
824 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 ...
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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 ...
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1answer
100 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 ...
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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 ...
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2answers
96 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 ...
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1answer
216 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}{...
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37 views

Find the date that sequencing was performed

I've got a bunch of RNAseq datasets from ENCODE and I'm looking to identify variables contributing to variance among my samples. For example, is there any easy way to find out the precise date which ...
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1answer
94 views

Tumor sample and heterozygous SNVs: what is the genotype of the normal cells?

In the following picture, you can see that in normal cells (yellow) we have two copies of each gene, while in tumor cells (violet), we just have one copy due to a clonal mono-allelic deletion. I do ...
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2answers
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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 ...
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1answer
83 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 ...
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1answer
268 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 ...
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1answer
63 views

Help Adjusting Bedtools Output in R

I have been asked to write a script in R to edit, using data.frame, this .fa file that is the result from extracting gene sequences from a repeat masker coordinate file on the genome I am working with....
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30 views

Which software is recommended for sequencing primers selection?

I have a list of primers for sequencing fungal ITS1/2 regions. I also have access to different fungal databases. I would like to perform in silico PCR analysis for comparing different primer pairs and ...
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1answer
117 views

Is it OK to use Blast+ to query NCBI's 16s rRNA database with 16s DNA sequences?

I have 16S DNA sequences from diversity sequencing, and want to query these against NCBI's 16S rRNA database. If I ultimately want to get taxids for the species that my sequences have, does it make a ...
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1answer
96 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 ...
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1answer
66 views

How to QC overamplified shotgun library?

I made some NEB ultra II whole-genome shotgun libraries with around ~200ng of template going into the indexing PCR step. The PCR was run for 13 cycles and I ended up having high library concentrations ...
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2answers
983 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 ...
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1answer
269 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 ...
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1answer
790 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 ...
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2answers
620 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 ...
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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 ...
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2answers
249 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, ...
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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: ...
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1answer
309 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 ...
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5k 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? ...
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220 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 ...
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1answer
66 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 ...
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1answer
120 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 ...
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1answer
33 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.
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341 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: ...
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96 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 ...
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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 ...
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3k 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 ...