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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60 views

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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1answer
44 views

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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1answer
17 views

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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1answer
36 views

Detecting SARS-CoV-2 variants from the mixed virus population

I have a fastq file from Nanopore sequencing data that contains reads from the UK and South Africa variants of SARS-CoV-2. These variants are identified by three key mutations in the receptor-binding ...
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1answer
64 views

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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1answer
20 views

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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1answer
408 views

What is "unmapped read segments" in the output of samtools idxstats?

samtools idxstats produces a four column output (see here) ...
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1answer
36 views

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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22 views

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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1answer
61 views

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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2answers
155 views

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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48 views

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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2answers
801 views

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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46 views

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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2answers
113 views

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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1answer
74 views

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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1answer
4k views

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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1answer
33 views

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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1answer
109 views

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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3answers
160 views

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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1answer
76 views

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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1answer
202 views

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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2answers
730 views

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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1answer
214 views

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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1answer
610 views

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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1answer
360 views

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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1answer
141 views

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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3answers
1k views

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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1answer
308 views

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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1answer
210 views

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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1answer
1k views

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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2answers
101 views

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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1answer
1k views

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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2answers
171 views

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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3answers
3k views

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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2answers
2k views

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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12answers
2k views

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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2answers
630 views

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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2answers
2k views

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 ...