Questions tagged [ngs]

use for general applications of high-throughput nucleotide sequencing methods that use short-read technology (e.g. Illumina, IonTorrent). For long-read sequencing, use 'long-read', or a more specific tag if applicable (e.g. nanopore, pacbio).

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0answers
21 views

Template files for bioattributes and meta-data

I am in process of submiting my FASTQ files to the SRA database. I am a little confused about how to fill the biosample attributes and SRA metadata. I would really appreciate if you anyone could share ...
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1answer
19 views

Trimmomatic trims most of the reverse strand

I have a paired-end data. It is from small part of human genome not a WGS or WES. I use BWA for alignment and do variant calling. Since I had some false positives I wanted to do trimming. I used ...
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1answer
34 views

Inheritance annotation for nsSNPs

I am relatively new to SNP analysis. Is there a database to find annotation about the Mendelian inheritance of SNPs? I have a small list of nsSNPs (non-synonymous SNPs) and I need to find how they are ...
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1answer
85 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........... ...
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2answers
46 views

Error after trimming illumina adapters

I am removing illumina adapters of the NGS data with a loop. My NGS data is storage in /data/HTS_seq/. I used this function: ...
0
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1answer
32 views

Question: Bismark methylation for non-uniquely mapping BS-seq reads

I am looking for the DNA methylation, specifically inside small RNAs; piRNA. I am mapping BS-seq reads on the genome with bismark. From the user guide of bismark, it maps reads uniquely on the genome, ...
0
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1answer
45 views

snakemake - using output file not explicitly declared as input for subsequent rule

I am writing a snakemake that, up to now should use raw sequencing reads to produce a draft assembly using canu use minimap2 to align the raw reads against the draft assembly, generated by canu. ...
0
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1answer
22 views

How can I extract all known mutations of my BAM (or SNP/INDL files)?

I am using a Genome Explorer tool to see all the mutations on my own DNA. My particular interest is on listing the variants and get their names/ids. Here are a few screenshots: You can see that ...
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1answer
87 views

Blast results filters

I performed a blastn search of NGS data against ssRNA database download from Internet, with a expected value 10-4. The size of NGS data reads is of 125 bp. I have analyzed the blast results of the ...
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2answers
43 views

How to remove sequence reads contains more than 2 X from multifasta file?

I have 5000 protein sequences in one multifasta file. I found more reads have gaps as X in their reads. So, want to eliminate those reads completely (Whole protein seq) from the file. I am keeping ...
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1answer
44 views

No MQ tags in VCF files

To call minority variants in my Mtb sequences I'm using a pipeline of ...
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1answer
24 views

How can I find the position of every mutation where the Allele Frequency is greater than X, in regards to a reference such as Hg19?

I have a Human genome sequenced in a BAM file (along with other files with the indels, snps, cnvs). I want to find every mutation with regards to the reference Human genome. However the majority of "...
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2answers
95 views

Generate VCF from different .bam files with different chromosome names

I have two resources of .bam files. One is generated by our lab (1 sample = 1 bam). One is downloaded online (again 1 sample = 1 bam). For the downloaded samples the chromosomes are labelled: chr1, ...
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1answer
73 views

How to get the intersection of peaks after peak calling using MACS2?

I have following 5 narrow peak files after peak calling. K14_peaks.narrowPeak K15_peaks.narrowPeak K16_peaks.narrowPeak K3_peaks.narrowPeak K8_peaks.narrowPeak I ...
0
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1answer
28 views

Downloading dataset from SRA (SOLiD Platform)

Trying to download colorspace data from SRA, but getting an error abi-dump -A SRR1657115.sra abi-dump.2.9.6 err: item not found while constructing within ...
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0answers
26 views

What kind of error-correction code is used in the error-correction code DNA sequencing?

In 2017, Chen et al reported a DNA sequencing technology called error-correction code (ECC) sequencing [1]. They borrowed ideas from communication and coding theory and encoded information redundency ...
2
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1answer
107 views

Any user friendly way to find rare mutations in whole genome raw?

Is there any user friendly way to find rare mutations in the individual human whole genome sequencing raw data? (from Dante, 30x coverage). To be more specific, I want to find mutations from this ...
0
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1answer
36 views

Add new tool to galaxy

I am trying to include a new tool to the galaxy main menu, following these instructions. tool_conf.xml doesn't exists only ...
3
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1answer
153 views

How can I export a full alignment from IGV as an image?

The IGV browser lets you export an alignment as an image (File => Save Image). However, this image only contains those reads that fit in the viewing window: As you can see in the image above by ...
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2answers
294 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 ...
2
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1answer
234 views

How to get a MSA fasta from BAM/SAM?

I am working with paired end NGS miseq data from a viral genome with multiple timepoints. I have filtered and trimmed this data for quality and adapter sequences. I have then merged the filtered ...
5
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1answer
722 views

PCA vs tSNE in single cell RNA-seq

What makes tSNE being the preferred dimensional reduction for visualization in single cell RNA-seq over PCA? I am aware that tSNE works better at showing local structures and fails to capture global ...
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2answers
341 views

Can we use GTEx data as control data for TCGA data?

I am using Recount2 TCGA data and was wondering is it right to use GTEx data as control data for this. I would really appreciate your views on this?
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5answers
241 views

Testing for viral/bacterial infection FASTQ files

What's the best way to test if a pair of Illumina FASTQ files from blood DNA extraction contain any reads from viral/bacterial infection from DNA in human blood? Thanks in advance. EDIT: Looking at ...
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2answers
132 views

VEP output SIFT_score unclear

We have been experimenting with VEP (Variant Effect Predictor). One of the meta data attributes that we are interested in is the SIFT score, indeed when we apply the dbNSFP plug we get a column ...
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0answers
82 views

How snippy makes MSA-like aligned fasta output from multiple samples?

From the log file it seems snippy doesn't do assembly. It only does mapping: fastq --> SAM --> BAM --> VCF --> consensus_seq/snps But if multiple ...
3
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3answers
180 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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0answers
83 views

amplicon sequencing set up

I have .fastq files from a targeted amplicon sequencing by Illumina MiSeq run. I want to get the target sequences to perform Blast search on them. What is the pipeline to use for processing amplicon ...
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2answers
1k views

How to count the number of mapped read in 100-bp window from a BAM/SAM file

Although I know how to get total number of mapped read using samtools flagstat (samtools flagstat file_sorted.bam) but I want to count total number of mapped read ...
4
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1answer
87 views

Simulate and test CNV workflow?

I'd like to evaluate a CNV project. My aim is to evaluate if the scripts are sufficient for calling reasonable CNVs. I know they have a paper, but their scripts may be buggy... and all papers are ...
3
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1answer
316 views

Why is the total read number still more than the paired in sequencing after removing the duplicate in samtools flagstat output?

After alignment using BWA, I have removed the dupliment using the samtools(Version: 1.9). My procedure is as follows: ...
2
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0answers
41 views

Calculating alignment/mapping time

I am trying to assemble a plant genome using AWS resources using velvet. Plant genome is huge (> 10 times human genome) and coverage is around 30 x. We are planning for de novo assembly with Velvet (...
3
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0answers
348 views

Fastest way to demultiplex bam file based on field

I have a bam file with aligned reads from multiple plates. For each plate I have a portion of the sequence identifier, thus for example ...
6
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5answers
936 views

Delete all 4 lines of a fastq read from a fastq file using read ID

I have the following error when running bowtie2: Error: Read HWI-D00466:116:CC62WANXX:3:1102:7363:63646 1:N:0:GCACACG has more read characters than quality values. I now want to remove all 4 ...
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1answer
774 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 ...
5
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1answer
160 views

vcftools: indel size histogram command returns empty file

I would like to get some summary statistics on a vcf file from one individual, which has over a million variant calls. I've tried to make a histogram of indel sizes with this command, ...
2
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1answer
198 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
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1answer
105 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 ...
0
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1answer
246 views

Fastqc- Per Base Sequence Quality

I am trying to figure out how to interpret the "Per Base Sequence Quality"? What does Position in reads (bp) mean? Also in order to draw this box-plot graphics, more than one quality scores are needed ...
4
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1answer
138 views

Efficiently aligning a lot of reads on the same small reference sequence

The context: I have a DNA-sequence coding for a protein, about 1500 bp in length. Using NGS, a lot of reads of (mutants of) this same sequence were acquired. All of these reads need to be aligned to ...
8
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1answer
88 views

Best practices for deciding if two structural variants are actually the same variant?

I know that I can use bcftools isec to compare two VCF files containing single nucleotide variants (SNVs); it will generate four possible output files: one with all ...
5
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2answers
101 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 ...
2
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3answers
1k views

Annotation with Prokka or RAST.

I was experimenting Prokka and RAST annotation tools. So, I took a well-annotated swinepox virus genome from genebank (NCBI Reference Sequence: NC_003389.1). I ran those sequences on Prokka and RAST ...
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3answers
176 views

Genome scaffolding

I assembled a virus genome using Ray and got around 5000 contigs. Now I want to scaffold those contigs to get a full genome (I am aware of the fact that there might be some gaps). I found one tool ...
0
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1answer
96 views

Using sickle for quality trimming

I am using sickle package from bioconda to trim my reads from the Illumina Miseq. When I tried following code, it failed to run ...
3
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1answer
222 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}{...
5
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2answers
4k views

Difference between samtools mark duplicates and samtools remove duplicates?

What is difference between samtools mark duplicates and remove duplicates ? Is it necessary to mark duplicates before removing duplicates with samtools?
6
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2answers
198 views

Meaning of the FORMAT fields of the VCF file coming from GIAB project

After reading the GIAB paper in https://www.biorxiv.org/content/early/2018/05/25/281006 and its Figure 1, I am still having trouble understanding the data inside the GIAB VCF file for HG001 (...
3
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3answers
179 views

Relationship between sequencing lane and ngs dataset

I am fairly new to NGS data analysis and I am struggling to understand the exact relationship between a sequencing lane and an NGS dataset. I should add I don't work in the lab, I only do ...
3
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2answers
266 views

Protein sequence from patient data

Currently, I am working on NGS data and my aim is to get significance prediction of variants present in the vcf file. As we know about SIFT Score for significance score prediction, I am trying to ...