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Non-uniform p-value distribution - help

I am running regressions using limma on 450k array DNA methylation data. I'm interested in the relationship between circulating levels of a hormone and DNAm in ~144,000 CpG sites I've preselected based on variability (non variable sites likely won't tell us anything about our variable of interest). I run regressions using limma, and plot the...

Demultiplex Novaseq run with mixed samples containing either UDI alone or UMI-UDI

I have the raw basecall files from a Novaseq run that was run using the XP workflow. In each of the lanes I have 8 samples with UDI only and 8 samples with xGen UMI-UDI. I need to demultiplex the samples and generate fastq files. The command I am trying to use is /root/bin/bcl2fastq --input-dir /pathtoNovaseqRun/Data/Intensities/BaseCalls \...

Create groups for PLINk comparisons

Hello all, I'm trying to analyse SNP array and since this is new to me I'm having difficulties. This array is the new Calvin CEL files from axiom. I had to use the axiom suite in order to get the SNPs. I got my data in VCF format and then was trying to use plink to do some statistical analysis. However, I found out that the array was conducted in humans...

scRNA gene expression without control/comparator

Hi everyone, I recently had a patient with a very rare lymphoma type and was able to perform scRNA-Seq of a fresh lymph node biopsy. Is there a way to check for differential gene expression based on one single sample, as I do not have any further (non-lymphoma) material from this patient to compare with his lymphoma sample? I am grateful for any...

merging multiple vcfs by column in Picard

Hello everyone, I used `picard MergeVcfs` to combine individual vcf files with the following command: java -jar /picard.jar MergeVcfs -I input.vcf -I input2.vcf -I inputs.vcf O= output.vcf.gz The resulting output file looks like this: #CHROM POS ID REF ALT QUAL FILTER INFO FORMAT 20 Chromosome_1...

pulling out TCR data

Hello! I have successfully loaded my VDJ data into my seurate object. Once I have identified the cluster of CD4 and CD8 I want to be able to pull out all the VDJ barcodes, sequence, clonotype, etc. I saw that WhichCells() command lets you pick out cells that have a certain experssion, but when I do this I only get 3 results returned (there is a lot...

Errors with Picard CollectRnaSeqMetrics

Hello, I'm trying to run a QC on my bam files for an RNAseq, but when I run the command: find *U*.bam -exec echo java -jar ~/path/picard.jar CollectRnaSeqMetrics I={} O=picard/{}.RNA_Metrics REF_FLAT=$RNA_HOME/refs/sacCer3.ncbiRefSeq.ref_flat.txt STRAND=SECOND_READ_TRANSCRIPTION_STRAND RIBOSOMAL_INTERVALS=$RNA_HOME/refs/ref_ribosome.interval_list...

Running PHATE on Seurat Object

I have single cell data from one sample that I am trying to run through the Seurat pipeline. After having obtained the UMAP plot, I am trying to compare it to the PHATE plot. However, I can't seem to run PHATE on a Seurat object. What are some ways I can go about this? Can I convert my Seurat object to some other data type and run the function on it?...

Can I add more than one effect (example: treatment and sex) to cuffdiff ?

I am having a problem to analyze the differentially expressed genes by using cuffdiff , because besides the two treatments I also have samples of different sex, and I would like to distinguish beteween them ? Is it possible to run a 2 step approach using cuffdiff (first with sex effect and repeat with treatemnt) ? If so , which files should I load...

What stops clusters from merging during bridge amplification?

Hello Everyone! I'm sorry if this is answered somewhere else, If it is please direct me to it. I cant seem to find much information. I'm wondering how DNA fragment clusters dont get all mushed together on a flow cell when sequencing after bridge amplification. It is my understanding that during RNA-Seq, RNA is converted to DNA, then the DNA...

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