Questions tagged [sequencing]

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

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55 views

Adapter trimming

I am trying to do adapter trimming, alignment and sorting for a range of large scale paired end fastq files. The code I am using is given below: ...
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What data set was used Gene Expression Data of Postmortem Tissues?

I want to run the experiments "Sample Application to Genotype-Tissue Expression (GTEx) Project Gene Expression Data of Postmortem Tissues" mentioned in "Enter the Matrix: Factorization ...
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Lower case vs. upper case nucleotids in sequence vs. dots at the end

What is the difference between lower case and upper case nucleotides in a sequence? My other question is what are the dots at the end of the sequence? Some examples are shown below: GGgG,GGgG,GGgG,...
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Can multiplexing in Sequel II SMRTcells reduce the coverage?

I would like to have high depth of coverage of the transcriptome, but I need to analyze several tissues. I was suggested to pool all 5 tissue samples in same SMRT 8M cell. Will this result in a ...
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How can Iso-Seq reverse transcriptase artifacts be avoided?

My end goal is annotate a de-novo assembled genome. When trying to select the best method for transcriptome assembly I read Iso-seq was the preferred method. However other people suggested Nanopore as ...
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What is the most accurate approach for de Novo sequencing?

I'm trying to decide between PacBio HiFi or Illumina sequencing platforms for sequencing the genome of aChrysina scarab. We want to identify the color pattern loci and also perform a phylogenetic ...
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Can we do NGS library preparation using UMI with large amount of DNA input?

We know that in next-generation sequencing (NGS), the unique molecular identifier (UMI) can reduce or eliminate sequencing or PCR errors and result in very high accurate data. Therefore, UMI is widely ...
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How can I generate ASV file from nanopore sequencing data?

I am converting the fast5 file to fastq by using guppy basecaller after that by using kraken2 classified the sequence. Now I am trying to generate ASV file. Is this possible to generate ASV file from ...
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Extracting base sequences from ABI/AB1 sanger sequencing chromatogram

I am trying to understand the sanger sequencing ABI/AB1 file format better, and extract base calls from given signal intensities over time. As I understand, reading in a raw AB1/ABI file into python, ...
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Does the number of RNA reads per cell obtained from the 10X scRNA experiment depend on amount of mRNA in given cell?

As we know, the amount of RNA reads per cell obtained from 10X scRNA experiment vary between cells. I wonder if this is effect of technical issues or does the number of RNA reads per cell obtained ...
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Interpreting 'samtools mpileup' output for multiple inputs

I would like to calculate sequencing coverage for a WGS project. Both long and short reads. I've used samtools as following: ...
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Why must a maximal non-branching path be a contig?

The following is from Bioinformatics Algorithms: Fortunately, we can derive contigs from the de Bruijn graph. A path in a graph is called non-branching if in(v) = out(v) = 1 for each intermediate ...
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Locate the saved template of minknow

I am using minknow on ubuntu. After saving the template (settings), where is that file locally stored in the system? Also what is its format/extension?
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What are Dummy fastqs?

I had to do a file transfer for in our system to run from fastqs and I came across the term "dummy fastq", not sure what exactly it means and what is the purpose of it in the workflow. Can ...
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How to retrieve fasta sequence after local blast?

I have created a Blast database using a reference genome. Then, I have performed a local blast search in command line using a gene of interest. I have obtained some hits with the usual Blasting ...
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Drug-DNA complex simulation using AMBER

I want to carry out the simulation of drug-dna complex by placing the drug molecule at particular site in my DNA sequence (intercalating site). I have PDB files of both drug and DNA sequence. I will ...
2 votes
1 answer
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When using cutadapt, can I specify an R2 adapter as optional when I have a required R1 adapter?

I'm using cutadapt 3.5 to trim adapters and perform some filtering on paired-end data. Both R1 and R2 sequences have 3' adapters that might be found depending on the sequence length, but R1 also has ...
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Shotgun analysis: Where and how to start?

I am a complete rookie in shotgun analysis, having only performed marker gene analysis before. I have a pipeline that I want to try but I can't wrap my head around how it all works. Please correct me ...
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How to Read SCF file in Python?

Is there any way that I can read SCF file in python like in R using sangerseqR, I have tried with Biopython, it seems it does not support this format.
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Where to find publicly available Sanger chromatography data?

I am looking for any public available databases where I can download Sanger chromatography data ideally in Ab1 or SCF file format. I need extremely large amounts of this, since I want to use it for ...
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2 votes
1 answer
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Low pass sequencing has been reported to detect common variants. How low can one go and get reliable data? Is 2X pass sequencing analysis possible?

I would like to use low pass sequencing to replace a genotyping chip to be able to detect variants up to 0.1 % allele frequency in available population data. What is the minimum depth I can opt for to ...
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Illumina vs PacBio vs Nanopore price

Can someone help me with comparing the cost per run of Illumina, PacBio and Nanopore sequencing technologies?
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Which format is the most raw format

I wanted information regarding which format is considered the most raw from an illumina sequencer ? fasta fastq bcl bam As per my research it should be bcl but I am not sure.
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Which sequencing technologies are considered short read

I couldn't find any information on this online. Which of the following sequencing technologies are considered short read technologies? Illumina, Oxford Nanopore, Ion Torrent, Pacific Biosciences
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Construct the Overlap Graph of a Collection of k-mers

I am doing a course on graph algorithms in genome sequencing and the current assignment involves building an overlap graph from a bunch of k-mers. Formally the problem is as follows: Input: A ...
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1 answer
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What is the right way of calculating a Phred score by hand?

i am trying to calculate mean Phred scores for my sequencing data, but i feel not very comfortable about it. There are actually two ways of calculating. (I just use an existing sample) giving: 3 reads ...
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Get frequency of index sequences of fastq file

I recently did some sequencing on a MiniSeq and unexpectedly the Undetermined_S0_L001_R1_001.fastq.gz file contained quite a lot of data (about 40% of total). I ...
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Modeling number of reads mapped to a gene

I am looking for a probability distribution of a number of reads mapped to a particular gene in metagenomic sequencing (NGS, shotgun, likely illumina). Naively one could model it via a binomial (or ...
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2 answers
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Filter rows of VCF file for Match=EXACT?

How do I remove rows in a VCF file on 1kGenome column, where Match=EXACT using bash ?
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What is a PacBio "movie file"?

I came across references to "movie files" from PacBio sequencing in this paper: https://www.jimmunol.org/content/204/12/3434 Specifically: Movie files used to generate results presented in ...
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How to determine a correct promoter sequence? (Bisulfite sequencing)

I'd like to ask you for help with an university assignment. First let me post the entire question: Design primers for bisulfite sequencing of the promoter of the ACE2 gene in microbat Myotis ...
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1 answer
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Quast duplication ratio and mismatches percent

When analyzing Quast results it seems that it doesn't calculate mismatches and indels in a useful way if the "Duplication ratio" is over 1. For example, that's what I get for an assembly ...
6 votes
1 answer
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How to search for high coverage SRA entries

The Question I want to find high coverage SRA entries, e.g., above 100x. I guess the best way is to use https://www.ncbi.nlm.nih.gov/sra with an appropriate search term. I don't mind if the search ...
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1 answer
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Metagenome simulation from a concatenated FASTA file

I am trying to simulate metagenome sequencing. So I will start with a file with a lot of concatenated genomes. From there, I would like to randomly extract 10,000 sequences of length 200bps. I got ...
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1 answer
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chipseeker annotation issue

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DNA sequence error annotation

Suppose I generate a DNA sequence of the following pattern. AAGTC And after being passed through a channel with insertion, deletion and substitution errors obtain the following sequence AAAGGC Where ...
2 votes
2 answers
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How can I subset WGS data to the level of WES variants?

I would like to compare mutational signatures1 in patients from different studies, however some studies are based on exome seq (i.e. ~20,000 coding variants) and some are from whole genome seq (i.e. ~...
2 votes
2 answers
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Are sequencing reads written differently when inserts and indexes are sequenced seperately?

I was going over different sequencing library protocols and noticed that the adapter sequence can vary between protocols. In some adapter sequences the index sits between the primer binding site and ...
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Fastq dataset after clustering

I'm doing a project for DNA archival storage systems Is there an open-source dataset used as ground truth for clustering? E.g A file containing the ground truth strand sequences, which have been ...
3 votes
2 answers
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Virus genome: protein inside another protein

The genome sequence for the GenBank virus JX869059.2 (a human betacoronavirus) contains, among others, two proteins: N protein (range 28566..29807) and Orf8b (range 28762..29100). The interval for ...
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Bacterial DNA at the tail of transcriptome reads. What does that mean?

I am assembling a transcriptome obtained from the Internet. The transcriptome was extracted from a human cancer tissue that had been previously grafted into a mouse. I have detected that many ...
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1 answer
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How to anonymise a bam file

I have a bam file of, for instance, RNA-Seq, which contains patient-identifiable data in the form of Single Nucleotide Polymorphisms (SNPs) throughout. I would like a technique to take this aligned ...
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How many markers or loci are used in forensic DNA profiling and the effect of the DNA sequencing?

I am reading the Wikipedia page on DNA profiling, specifically STR analysis. It says that DNA profiling relies on matching 20 or so loci. It also gives an account of finding false identification. But ...
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4 answers
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Separate multiple sequence into different file, one sequence per file

I have a file contain multiple sequence, and I want to separate them by "gene:" into different file. example: example.fa ...
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2 answers
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How to combine two Genome-wide Association Study (GWAS)? [closed]

I did a GWAS analysis in the past for antibiotic resistance of E. Coli and the results were interesting (matching the literature). I did a new GWAS analysis for some new samples, but the results are ...
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1 vote
1 answer
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merge the matrix from computematrix of deeptools

I have several matrix from computematrix of deeptools. I need to merge two of them using "computeMatrixOperations cbind -m input1.mat.gz input2.mat.gz -o output.mat.gz" but I am running to error "/...
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1 answer
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Assembly reads having a copy of their beginning in their tail

I am analyzing the reads for the SARS-CoV-2 assembly with id SRR11140748. Apparently these reads were obtained with parallel sequencing by Illumina and Oxford Nanopore Technologies. I have found these ...
2 votes
1 answer
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What is the meaning of these misaligned reads in a sequencing run?

I am analyzing some SARS-CoV-2 sequencing runs abd often find read alignments like the one in the image. ...
2 votes
2 answers
872 views

Marking optical or PCR duplicates with picard vs. samtools flagstat

I am trying to identify the best library prep method for noninvasive prenatal test samples, to be sequenced with Illumina Novaseq. One metric that I am evaluating is the number/ percentage of PCR or ...
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1 answer
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Overlap in Paired-end Reads for Sequencing?

Regarding Sequencing: Do/can the paired-end reads have overlaps?
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