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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questions with no upvoted or accepted answers
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Low Fraction of usable antibody reads in CiteSeq
we performed a combined gene expression and CiteSeq experiment with the 10x VDJ kit and 20 conjugated antibodies and sequenced on hiseq. I used cellranger to process the sequencing output. The ...
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Can multiplexing in Sequel II SMRTcells reduce the coverage?
I would like to have high depth of coverage of the transcriptome, but I need to analyze several tissues. I was suggested to pool all 5 tissue samples in same SMRT 8M cell. Will this result in a ...
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A question on Homer normalization
When using annotatePeaks.pl script from Homer software to create histograms, the output is normalized per bp per peak (on top of normalization to 10 million tags).
What does it mean to normalize per ...
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649
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Fastest way to demultiplex bam file based on field
I have a bam file with aligned reads from multiple experiments. For each experiment I have a portion of the sequence identifier, thus for example
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Off-target % for whole-exome sequencing panel
My samples have been sequenced with the Twist Exome 2.0 sequencing panel, and I want to assess how efficient the sequencing has been. By efficient, I mean examining metrics such as the uniformity (...
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calculate mismatch frequency/rate from a BAM file
I am working with PAR-CLIP data where experimental procedure induces T to C mismatches. The other types of mismatches we can consider coming from artifact such as PCR errors. It could also be an ...
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78
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BAF and LRR calculation and usage
I'm working with a vcf file, in particular I'm trying to identify a gene duplication in a sequenced genome.
I studied bcftools cnv command, but for this tool is needed a vcf file containing a BAF and ...
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77
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Adapter trimming
I am trying to do adapter trimming, alignment and sorting for a range of large scale paired end fastq files. The code I am using is given below:
...
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What data set was used Gene Expression Data of Postmortem Tissues?
I want to run the experiments "Sample Application to Genotype-Tissue Expression (GTEx) Project Gene Expression Data of Postmortem Tissues" mentioned in "Enter the Matrix: Factorization ...
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176
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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 ...
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2
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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 (...
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DNASTAR viral-host integration assembly keeps failing
I have two NGS files from an NGS company corresponding to the sequencing data from a tumor sample as follows:
TB_7710391_R1.FASTQ.gz
TB_7710391_R2.FASTQ.gz
I have downloaded the genome for MCPyV as ...
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Is it common to get different number of SNVs+Indels across samples from vcf files generated using GATK and DRAGEN (counts are higher for GATK)?
We have 2 vcf (Whole Exome Sequencing (WES) data; germline samples) files (e.g., vcf_1 and vcf_2). vcf_1 was generated (Ref. genome: hg38) using the GATK pipeline for 250 children and their parents ...
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Lower case vs. upper case nucleotids in sequence vs. dots at the end
What is the difference between lower case and upper case nucleotides in a sequence? My other question is what are the dots at the end of the sequence? Some examples are shown below:
GGgG,GGgG,GGgG,...
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System specifications for NGS data analysis
I have a 3.7 GB whole genome data of a eukaryote, for which genome assembly, gene prediction, and annotation steps have to be performed. In some time I would also need to analyze the transcriptome ...
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How can I generate ASV file from nanopore sequencing data?
I am converting the fast5 file to fastq by using guppy basecaller after that by using kraken2 classified the sequence.
Now I am trying to generate ASV file.
Is this possible to generate ASV file from ...
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1
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1
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308
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Extracting base sequences from ABI/AB1 sanger sequencing chromatogram
I am trying to understand the sanger sequencing ABI/AB1 file format better, and extract base calls from given signal intensities over time.
As I understand, reading in a raw AB1/ABI file into python, ...
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Illumina vs PacBio vs Nanopore price
Can someone help me with comparing the cost per run of Illumina, PacBio and Nanopore sequencing technologies?
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Calling variants with DeepVariant on targeted NGS sequencing (custom library)
I am seeking advice regarding DeepVariant analysis.
To avoid false positives I'm using several variant callers and then the resulting common set will be considered as TP variants.
One of the callers ...
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1
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50
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Genomic relationship matrix explanation
I am seeking the definition of the genetic distance matrices used in genomic prediction.
What is the difference between a Rodger distance and kinship matrix?
what people mean when they talk about ...
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Fastq dataset after clustering
I'm doing a project for DNA archival storage systems
Is there an open-source dataset used as ground truth for clustering?
E.g
A file containing the ground truth strand sequences, which have been ...
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How many markers or loci are used in forensic DNA profiling and the effect of the DNA sequencing?
I am reading the Wikipedia page on DNA profiling, specifically STR analysis. It says that DNA profiling relies on matching 20 or so loci. It also gives an account of finding false identification. But ...
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108
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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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Find the date that sequencing was performed
Background I've obtained RNAseq datasets from ENCODE and I'm looking to identify external variables contributing to sequence variance among these samples.
External variables For example, clustering ...
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Which software is recommended for sequencing primers selection?
I have a list of primers for sequencing fungal ITS1/2 regions. I also have access to different fungal databases. I would like to perform in silico PCR analysis for comparing different primer pairs and ...
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I have two files: mys.nuc and ms.prep and need to use bioseq to extract the ORF sequences and save as “mys.nuc2”
Below are the two files I am working with:
...
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How do I build the MinusB database for Kraken2? (Taxonomy issues)
I am attempting to build my own custom database for Kraken2. I have two questions:
If I have the MCPyV genome in a file called MCPyV.fasta, how do I build a database with just this?
How do I build ...
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Kraken2 Standard Database failing to build (unexpected FTP path)
I am attempting to build just the standard Kraken2 Database by using the following command:
kraken2-build --standard --threads 24 --db $DBNAME
I am returned the ...
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73
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Locate the saved template of minknow
I am using minknow on ubuntu. After saving the template (settings), where is that file locally stored in the system?
Also what is its format/extension?
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Drug-DNA complex simulation using AMBER
I want to carry out the simulation of drug-dna complex by placing the drug molecule at particular site in my DNA sequence (intercalating site). I have PDB files of both drug and DNA sequence. I will ...
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99
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Why is bcl2fastq2 taking so long to calculate stats?
Our lab has been using bcl2fastq v2.20.0.422 to demultiplex RNA-seq data sequenced on an Illumina Novaseq machine on a beefy EC2 instance and we've run into the strange problem: namely that while ...
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272
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BWA-MEM2 alignment-Snakemake
I have started using snakemake 6.5.2 to align fastq files with reference file. I have pasted the error below in this question. How to allocate memory in the snakefile and read the header from samfile, ...
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DNA sequence error annotation
Suppose I generate a DNA sequence of the following pattern.
AAGTC
And after being passed through a channel with insertion, deletion and substitution errors obtain the following sequence
AAAGGC
Where ...
