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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Why do some assemblers require an odd-length kmer for the construction of de Bruijn graphs?
Why do some assemblers like SOAPdenovo2 or Velvet require an odd-length k-mer size for the construction of de Bruijn graph, while some other assemblers like ABySS are fine with even-length k-mers?
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Why does the FASTA sequence for coronavirus look like DNA, not RNA?
I'm looking at a genome sequence for 2019-nCoV on NCBI. The FASTA sequence looks like this:
...
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4
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Why sequence the human genome at 30x coverage?
A bit of a historical question on a number, 30 times coverage, that's become so familiar in the field: why do we sequence the human genome at 30x coverage?
My question has two parts:
Who came up ...
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1
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Is it possible for coronavirus or SARS to be synthetic?
I have heard several conspiracy theories regarding the origin of the new coronavirus, 2019-nCov. For example that the virus and/or SARS were produced in a laboratory or were some variant of Middle ...
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3
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How to deal with heterozygosity during polishing of genome assembly based on long reads?
All the long-read sequencing platforms are based on single-molecule sequencing which causes higher per-base error rates. For this reason a polishing step was added to genome assembly pipelines - ...
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How can we distinguish between true zero and dropout-zero counts in single-cell RNA-seq?
In single-cell RNA-seq data we have an inflated number of 0 (or near-zero) counts due to low mRNA capture rate and other inefficiencies.
How can we decide which genes are 0 due to gene dropout (lack ...
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16
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3
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Designing a lab NGS file database schema
I am the resident Bioinfo Geek in a hospital academic lab that routinely employs NGS as well as CyTOF and other large volume data producing technologies. I am sick of our current "protocol" for ...
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2
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How can I call structural variants (SVs) from pair-end short read resequencing data?
I have a reference genome and now I would like to call structural variants from Illumina pair-end whole genome resequencing data (insert size 700bp).
There are many tools for SV calls (I made an ...
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How to simulate NGS reads, controlling sequence coverage?
I have a FASTA file with 100+ sequences like this:
>Sequence1
GTGCCTATTGCTACTAAAA ...
>Sequence2
GCAATGCAAGGAAGTGATGGCGGAAATAGCGTTA
......
I also have a ...
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1
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Why is bwa-mem the standard algorithm when using bwa?
The industry standard for aligning short reads seems to be bwa-mem. However, in my tests I have seen that using bwa backtrack (bwa-aln + bwa-sampe + bwa-samse) performs better. It is slightly slower, ...
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How to make a distinction between the "classical" de Bruijn graph and the one described in NGS papers?
In Computer Science a De Bruijn graph has (1) m^n vertices representing all possible sequences of length n over ...
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Is it possible to perform MinION sequencing offline?
I vaguely remember, that the original plan of Oxford Nanopore was to provide cheap sequencers (MinION), but charge for base-calling. For that reason the base-calling was performed in the cloud, and ...
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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 <...
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What are the pros and cons of the different basecallers in Oxford Nanopore Technology Sequencing?
What are the pros and cons of the different basecallers in Oxford Nanopore Technology Sequencing?
I am about to start a MinION run on my laptop. What should I consider when choosing my basecaller? ...
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Which quality score encoding does PacBio use?
Do you know which quality score encoding PacBio uses now? I know some of their file formats have changed in the past year or two, but I haven't found much on their quality score encoding.
The most ...
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What is the index fastq file (sample_I*.fastq.gz) generated when demultiplexing Illumina paired-end runs?
What is the index fastq file that comes with some Illumina sequencing datasets? (The samplename_I*.fastq.gz file.)
For example, I recently received some 10X ...
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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 ...
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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 ...
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A new paper suggests the Corona Virus has "Uncanny similarity of unique inserts in the 2019-nCoV spike protein to HIV-1" - What does this mean?
Quote:
We found 4 insertions in the spike glycoprotein (S) which are unique to the 2019-nCoV and are not present in other coronaviruses. Importantly, amino acid residues in all the 4 inserts have ...
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Why does this human bam file only have one copy of each chromosome?
As we know that in human DNA sequence, one copy of chromosome comes from mother's DNA and another copy comes from father's DNA so as to form two copies of each chromosome in human DNA. So, if we ...
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How do you generate read-length vs read-quality plot for long-read sequencing data (e.g., MinION)?
How do you generate read-length vs read-quality plot (heat map with histograms in the margin) for long-read sequencing data from the Oxford Nanopore Technologies (ONT) MinION? The MinKNOW software ...
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2
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How to transfer gff annotations in genome with extensive duplications?
Microbial genomes can contain extensive duplications. Often we'd like to transfer annotations from an annotated species to one that is newly sequenced.
Existing tools (e.g. RATT, LiftOver, Kraken) ...
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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 ...
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1
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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 ...
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4
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Why Illumina if PacBio provides longer and better reads?
PacBio provides longer read length than Illumina's short-length reads. Longer reads offer better opportunity for genome assembly, structural variant calling. It is not worse than short reads for ...
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What is 'k' in sequencing?
When a DNA sequence is sequenced, I've only ever dealt with A,T,C,G and N which indicates un-identifiable bases. However, I came across a 'k' recently and I had asked another researcher who gave me an ...
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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 ...
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Is there a point in recalibration of scores for variant calling?
The most variant calling pipeline GATK include a Base Quality Score Recalibration (BQSR) which requires a list of known variants. Recently, some work has been done for reference-free recalibration of ...
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dog coordinates (canFam3) to human coordinates (hg19)
I've converted dog coordinates to human using UCSC LiftOver. These are 200bp intergenic regions that are differentially methylated from normal dogs to cancer dogs. I've converted these to human ...
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What is the difference between NGS, 2GS, SBS and HTS?
I've come across a bit of confusion about the initialism NGS, so think it would be a good idea to clarify this term (and similar terms like 2GS, SBS, and HTS) for this site with a bit of discussion.
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7
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1
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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:
...
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Reject reads with low quality bases from a Bam file through pysam
I have a code below:
...
7
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1
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What is a good pipeline for using public domain exomes as controls?
I'm currently attempting association analysis with an extremely small set of patient exomes (n=10), with no control or parental exomes available. Downloading the ExAC VCF of variant sites (http://exac....
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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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How can I edit a specific FASTQ read in place, given the read ID?
I am introducing SNVs into specific samples in order to estimate false negative rates for a variant calling pipeline. I know reads can be simulated but I would actually prefer to use the real data so ...
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Parallel processing of scripts that use obitools
I have an obitools script (de Barba et al. 2016) that I would like to run faster. How would you run it in parallel to cut down on time?
...
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2
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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 ...
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1
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checks for spike-in sequence controls
I would like to know what do people verify when designing/using spike-in controls, to be used in sequencing experiments (mainly Illumina). So far I came up with this list:
Does it align only to a ...
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1
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What's the scaling for HOMER metagenes?
I'm trying to use HOMER to make a metagene profile over gene bodies using a bedgraph file I've generated. The problem is that every time I do, I get really weird scaling on the y-axis. I should be ...
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2
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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 (...
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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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QC measures for NGS sequencing
What are good means for performing quality control (QC) or NGS reads?
I'm aware of:
FastQC
NGS Screen
Kraken (e.g., for screening against contaminants)
What are other popular means for such QC?
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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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6
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Identifying Indels from Chromatograms
I have around 100 chromatograms (.ab1 files) from Sanger sequencing a genome at loci believed to have an indel.
I'm new to interpreting this kind of data in ...
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Why do I get so many insertions from Minimap2 on my Nanopore WGS?
I'm a starting my analysis on nanopore whole genome sequencing. I start my analysis from this popular Github.
The sample I downloaded was WGS for NA12878, so I would assume it's alignment to GRCh38 ...
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5
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2
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Perfect Phylogeny vs Maximum parsimony
I am searching various sources about phylogenetics. I saw some materials about perfect phylogeny and also phylogenies acquired from maximum parsimony constraint. They seem very similar to me. Are they ...
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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?
5
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Can a data file in VCF format be converted into FASTA?
I'm considering purchasing the 'MyGenome' product by Veritas Genetics to analyze my genome for a project. I'd like the data to be in FASTA format, but Veritas only provides VCF data. Is it possible to ...
5
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2
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Can I use my computer while MinION is sequencing without negatively affecting the run?
I have a Mac Book Pro and I am about to start a sequencing run on the MinION. MinKNOW is going to be running for 2 days.
Can I use my computer during sequencing? Can I browse the web and use Excel, ...
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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 ...
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