Questions tagged [reads]
Reads are the sequences output by a sequencing machine after the raw signal (e.g. light, electricity) is converted into bases by a basecaller.
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Determining fragment mean and fragment stdev for MaSuRCA config file
Similar to this unanswered question on Biostars, I am using MaSuRCA for the first time and want to know how other MaSuRCA users are determining fragment mean and fragment stdev. My understanding is ...
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If fastp output is not a good measure of FASTQ correctness, what is?
In the beginning of my pipeline, I just fed the paired reads (2 files) into fastp, with the default options, and assumed it would do a good job preparing the reads for the next step: alignment
But I ...
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Comparison of fastq files reads
My goal is to compare reads from two different fastq files on a Linux machine.
The following are the comparisons to perform:
How many common reads are between the two fastq files?
How many reads are ...
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Filtering paired-end reads with sambamba: avoid discarding reads on the minus strand
I have a BAM file (DNA, shallow whole genome sequencing at ~1X) where I want to filter reads (using sambamba) to keep only those which have a template length > 20 and mapping quality > 20, ...
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Extract read names and the associated nucleotides on specific positions from a BAM file (in R)
Let's assume I have a BAM file and several positions that I would like to examine more closely in this alignment. My goal is to find out whether these positions are on the same reads and which ...
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Connection between Detected Genes and The Read Counts
I have been trying to understand the Seurat for analysing scRNA-seq data. It comes to my mind that the main data is organised in the Seurat object with rows as genes and columns as the cells, and the ...
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Combining read counts from three separate GEO studies
I want to do differential expression analysis with DESEQ2. I have three read counts files downloaded from GEO (small RNAseq based) where the number of miRNAs and id is nearly the same. These studies ...
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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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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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What is "unmapped read segments" in the output of samtools idxstats?
samtools idxstats produces a four column output (see here)
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How to extract reads with INDELs > a given size?
I'm trying to modify this https://www.biostars.org/p/253774/
To get reads with deletions > 20bp
I think this gives reads with exactly 20bp dels:
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How to control/normalize for number of reads when calling SNPs using RNA-Seq?
I used the GATK pipeline to call SNPs on males and females using RNA-Seq data. But the males have a higher read count (~43-46M reads) than the females (~40-42M reads). This causes SNP counts to be ...
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If a gene is expressed at a level of 1/1200 compared to the average gene, how is probability 50:50 that we have a read mapped to it?
I am reading a book about RNAseq analysis and it says
"To calculate the probability that a read will map to a specific gene, we can assume an average gene size of 4000 nt (100 M nt divided by 25,...
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Does rRNA depletion protocol give higher number of mapped reads in Intronic regions?
Recently, I have downloaded a publicly available dataset, which are 350 tumor samples. I see the following information from the published paper.
They used Ribo Zero Gold and rRNA was depleted. Strand ...
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Clarification about how mapping to a reference genome works in RNA seq
When mapping reads to a reference genome, how is it possible to tell which part of the genome the read is referencing if the genome possibly has the same repeated sequences in different areas? What I ...
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Extract reads from bam files by their @RG
How could I extract reads from bam files by their read groups @RG ?, I've got one file containing all reads for 5 samples, the SM is appears NONE, So, I want to extract each sample reads by the @RG ...
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How to obtain the extended unaligned bases of the read in reference based genome assembly?
I did illumina read mapping based on a particular gene sequence (reference sequence) using the following command,
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In-sample and across samples normalized expression
I want to get the expression data that is in-sample normalized like FPKM and also across samples normalized as obtained using DESeq2 or else.
What I am currently doing is that I first normalize the ...
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RNA-seq gene/transcript read counts database for Mouse
Is there any RNAseq gene/transcript read count database for the mouse? I already know about ARCHS4, looking for some other source.
Thank you
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Extracting all reads from bam file which match read IDs in another file
I have a long list of read IDs of interest to me in a file called read_names.txt. it is simply in the format:
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Does read sequence identifier need to be unique for proper alignment?
I am preprocessing a set of scTHSSeq reads in which each read sequence identifier has been replaced with the cell barcode, e.g.:
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Low percentage of reads with consistent barcodes in Split-Seq run
I am following this answer to select reads with allowed barcodes from a Split-Seq run. In particular, I have a whitelist for each round of barcodes (3 rounds) and I filter out reads which have more ...
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How to detect mismatch before mapping in RNA-Seq data
In the methods of this paper, the authors say:
Reads were then filtered based on quality score in the UMI region. Any read with >1 low-quality base (phred <=10) were discarded. Reads with more ...
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What is and how to detect a dephased read
In the methods of this paper, the authors say:
To simplify analysis, we first removed any dephased reads in our library (last 6 bases of read did not match the expected sequence).
I have read this ...
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Demultiplexing and preprocessing for custom single-nucleus Drop-seq data
I am trying to reproduce the preprocessing of paired-end sequencing reads in Lake et al. (ref. 1).
First, paired-end reads were filtered out if read 1 had more than four non-T bases in the last ten ...
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Reads mapped to exonic, intronic and intergenic regions
After the alignment step I checked the rnaseq metrics of all the samples. Among 40 samples three samples show high percentage of reads mapped to intronic regions. What could be the reason?
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How to get rid of the temp files while using "featureCounts" for extracting readcounts from bam files?
I used this command for extracting read counts for almost 60 samples using featureCounts.
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Select top 100 genes ranked by variance in read counts
I have a gene expression data (read counts) table with many samples. Showing some of them below:
...
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Counting repeated kmers sequences that match at least x % of reads sequence
Working on a fastQ file, I would like to get the occurrences of repeated sequences for all possible kmers of a given length that cover at least 90% of the read's length for the whole data set.
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While opening a Bam file with the SeqAn library I get 'seqan::FileOpenError' [closed]
When I try to open a simple bam file with the SeqAn library using the following code:
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Why does cutadapt remove low quality bases from the ends of reads only?
I use cutadapt to remove low quality bases from my Illumina reads. The algorithm only removes low quality bases from the end until it reaches a good quality base. If there is a bad quality base beyond ...
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Counting reads for each biotype
I have bam files. I want to find the total read counts associated with all the biotypes, eg snRNA,rRNA,tRNA mRNA,scRNA,snoRNA etc. I can use ht-seq count to get read counts for the genes, but is ...
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Scaling by linear regression against the number of reads
I am trying to build the preprocessing pipeline presented in The Tabula Muris Consortium et al. (pp).
It is a pipeline to preprocess single-cell sequencing data. There is one step that is not clear:
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How to remove all BAM read groups from all reads (not just the header)?
I have problem with one my BAMs---it appears to have invalid read groups.
Normally when I have such a problem, I remove all the read groups from the BAM header as follows:
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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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What is local realignment and what is the problem it solves?
I am trying to understand local realignment but I could not get a clear idea of what is the problem solved by it.
For example, reading Homer and Nelson (2010):
Because alignment algorithms map ...
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Genome assembly from error-prone reads
I understand how to assemble genome from error-free reads. I implemented like this:
Construct directed overlap graph with reads as vertices and edges as
maximum overlap between two vertices. ...
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How to check whether all BAM read contain defined read groups?
I'm trying to investigate whether there are errors within my BAM. After looking at the BAM header to see whether the read groups exists (using samtools, i.e.
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Filtering step for read counts data
I have around 1200 samples as columns and 60,000 genes with Htseq-Counts data. Before normalization with voom function I want to do filtering step.
I want to remove genes whose expression is == 0 in ...
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Random access on a FASTQ file
I would like to select a random record from a large set of n unaligned sequencing reads in log(n) time complexity (big O ...
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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, ...
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Difference between BWA-backtrack and BWA-MEM
Many of my colleagues recommend I use BWA-MEM instead of regular old BWA. The problem is I don't understand why and reading the BWA man page doesn't seem to help the matter.
What is the difference ...
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