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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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1answer
26 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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0answers
67 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 ...
0
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0answers
29 views

Subtyping Agglomerated Mutations By Annotation Relations

Subtyping Agglomerated Mutations By Annotation Relations [SAMBAR] is a method to de-sparsify somatic mutation data by summarising these data into pathway mutation scores. This method has originally ...
4
votes
1answer
351 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 ...
0
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2answers
89 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?
1
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5answers
171 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 ...
1
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2answers
79 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 ...
2
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0answers
47 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
votes
3answers
96 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 ...
1
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0answers
65 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 ...
2
votes
2answers
456 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
votes
1answer
62 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
votes
1answer
121 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
37 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 (...
6
votes
5answers
342 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 ...
1
vote
1answer
179 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 ...
2
votes
1answer
144 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
votes
1answer
83 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
votes
1answer
115 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
votes
1answer
77 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
73 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
votes
2answers
87 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
votes
3answers
493 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 ...
1
vote
3answers
104 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
51 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
votes
1answer
214 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
2k 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
votes
1answer
105 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
votes
3answers
94 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
votes
2answers
193 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 ...
1
vote
1answer
336 views

update dbSNP ID

I have used dbSNP build 138 to tag 'rs' ID in my VCF file. But later I came to know that now dbSNP build 150 has been released. I want to update the rs IDs in my VCF file. I tried but I found a ...
2
votes
1answer
196 views

Is there a publicly available tumor-normal sample?

I am looking for a publicly available matched tumor-normal sample. I need Illumina fastq reads (or an aligned bam file, since I could extract the reads from it) from a tumor and a matching, non-tumor ...
1
vote
2answers
40 views

Mapping bisulfite reads to a reduced size genome

Is it worth mapping bisulfite reads (WGBS) to a reduced size genome? I'm interested only in modifications in CpG context, thus instead of mapping to a whole genome (human genome) I would: Extract ...
4
votes
2answers
1k views

How can I count the number of reads that support a variant in a bam file?

I am calling variants from a human sample using bwa mem to align the reads and gatk to call the variants. I'm trying to understand why a specific variant was not called in my sample. I have checked ...
2
votes
2answers
240 views

Joining scaffolds to get full genome?

I am using Ray to assemble a about 7.2 kb picornavirus genome. I am using k-mer of 55. Following is the exact code that I used. ...
1
vote
2answers
63 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 ...
3
votes
1answer
251 views

problem of “ordering in physical positions” phasing SNPs with Shapeit

I'm trying to phase my data (whole genome resequencing SNPs) with Shapeit (from ped file) and I get this error message : ...
7
votes
2answers
2k 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 ...
0
votes
1answer
368 views

How to proceed after contigs.fa file obtained from VELVET assembly

I want to assemble a linear genome and I have a reference genome too. I ran the following commands for de novo velvet assembly: ...
6
votes
3answers
662 views

What is deep sequencing?

People talk about deep sequencing. Is there any way to calculate how deep the sequencing is ? What should be the optimum depth to get reliable data ? I am doing whole genome sequencing of a virus ...
2
votes
1answer
183 views

Why are inversions defined as the reverse complement and not just the reverse of the reference?

I can’t quite understand the way inversions are defined. In particular I expect an inversion to be only a reversed version of the reference and not its reverse complement. Most sources use diagrams ...
5
votes
1answer
1k views

hg38 GTF file with RefSeq annotations

I'm not sure what I'm missing, but I'm struggling to find an official hg38 GTF file with RefSeq annotations. I'd like to provide ...
2
votes
1answer
573 views

VerifyBamID freemix score

We all have used freemix score at some stage to check contamination or swaps in our sequencing experiment, but can anyone once for all explain how the score is calculated?
6
votes
2answers
383 views

How can I edit a specific FASTQ read in place, given the read ID?

Given a read ID, I want to edit a single basecall (e.g. the 12th base) for just that read from within a large FASTQ file containing millions of reads. example, i want to change the 12th base ('C') in ...
8
votes
1answer
578 views

FASTQC overrepresented sequences after trimming

I have a set of RNA-seq samples from different experiments (Single and Paired End, depending on the experiment). I ran FASTQC in all the samples and found overrepresented adapter sequences: I removed ...
4
votes
1answer
60 views

Best way to detect long insertions in bisulfite sequencing data?

I am interested in identifying indels in whole genome bisulfite sequencing data (76bp paired end). Currently, I do this by setting the -rfg and ...
7
votes
1answer
298 views

Reject reads with low quality bases from a Bam file through pysam

I have a code below: ...
3
votes
1answer
120 views

What sequencing data metrics should I record?

I'm new to bioinformatics and Im starting a new microbial sequencing project and I'd like to record all of the qc data correctly. This is research and I'm not sure what I'll need later. Is there any ...
7
votes
1answer
362 views

How to simulate “base error rate” in art_illumina?

I'd like to simulate 10% sequencing error using art_illumina. The simulator doesn't have a parameter that I can just give the 10%, but it has this: ...
10
votes
7answers
5k views

Fast way to count number of reads and number of bases in a fastq file?

I am looking for a tool, preferably written in C or C++, that can quickly and efficiently count the number of reads and the number of bases in a compressed fastq file. I am currently doing this using <...