BTEP maintains several Question and Answer Forums of interest to the NCI/CCR community.
Currently, there are forums on these topics listed below:
If you wish to ask a question go to the Ask Question Page and submit your question.
a few experimental design-related questions on single cell RNA-seq?
Categories: Single-Cell RNA-Seq
Views: 2426 Asked By:- We are planning to multiplex ~8 solid tissue samples (normal human lung) for scRNA-seq using natural barcodes and we have a few experimental design-related questions.
- The main optimization we need is to keep the integrity of all the cell types in the tissue throughout fresh tissue dissociation-freezing-thawing-multiplexing procedure. We are concerned that epithelial cell types (the type that we are most interested in) are especially vulnerable to freeze-thawing process. What would you recommend to optimize to achieve this goal? For example, we are looking into adjusting the enzyme content/concentration to make the dissociation step less harsh to epithelial populations, as well as testing different freezing media.
- To achieve the cell viability of cryopreserved cells suitable for 10X 3’ sequencing, would it be worth comparing commercial dead cell removal kit vs. FACS sorting directly into 10X library for removing dead cells after thawing?
- What considerations should go into pilot testing for an efficient way to process 8 samples at the same time to minimize cell loss during the potentially long hands-on time?
- We are aiming to multiplex 8 samples based on the feasibility stated in the literature, but do you have experience on how feasible it is in reality? E.g. should we consider testing 4 samples to maximize the success in sample identify deconvolution?
- We are planning of a pilot experiment of 10X 3’ RNA for a couple of 8-plexed reaction before we expand onto larger samples. In addition to the standard QC metrics (e.g. viability, number of detected cells and genes per cell, mitochondrial content etc) what would you recommend to pay attention to to determine the quality of the data?
- In general, validity of cell clusters based on the gene expression might be hard to assess. What are the methods that you recommend to validate the quality of our final results? (e.g. mapping to public clustering data of the same tissue type, matching to Flow cytometry data for select markers, CITE-seq for a limited antibody profile)
Administrator issues with PC government computer
Categories: BIOSTAR
Views: 1966 Asked By:How will I be able to install the programs on the PC government computer?
Analyzing Short/Long read sequencing results and interpreting splicing events.
Views: 4729 Asked By:Asking before deleting
Categories: BIOSTAR
Views: 868 Asked By:Hi, during the last session the instructor showed that her Terminal would confirm before deleting a directory or file. I thought the instructions for how to set up our environment to do the same were going to be posted here.
Thanks for your help
Diana
BTEP R course help sessions
Categories: R
Views: 25 Asked By:BTEP R Courses
Categories: R
Views: 27 Asked By:Can we have a brief introduction of types of data in Bioinformatics?
Views: 4109 Asked By:Can you give some suggestions cell identity annoation and recommend packages good at cell identity annotation? What is the most used practice, manually or auto?
Categories: Single-Cell RNA-Seq
Views: 5784 Asked By:Can you recover a removed file after issuing the rm command?
Categories: BIOSTAR
Views: 1890 Asked By:Can you recover a removed file after issuing the rm command?
Can you say something about trajectory analysis vs. velocity analysis?
Categories: Single-Cell RNA-Seq
Views: 11577 Asked By:ChIP-seq versus other sequencing methods: What are the advantages and disadvantages of using ChIP-seq instead of ATAC-seq?
Categories: ChIP-Seq Data Analysis
Views: 2644 Asked By:ChIP-seq versus other sequencing methods: What is CUT & RUN? How do you compare ChIP-seq and CUT & RUN?
Categories: ChIP-Seq Data Analysis
Views: 2914 Asked By:Comparing to outside sources of data: Do you have recommendations for frameworks or programs for integrating ChIP-seq and RNA-seq or microarray data?
Categories: ChIP-Seq Data Analysis
Views: 1823 Asked By:Comparing to outside sources of data: What is a good website to check for transcription factor binding motifs and how they regulate expression?
Categories: ChIP-Seq Data Analysis
Views: 1904 Asked By:What is a good website to check for transcription factor binding motifs and how they regulate expression?
Control Types: Can we use any DNA-seq as an input sample?
Categories: ChIP-Seq Data Analysis
Views: 1036 Asked By:Can we use any DNA-seq as an input sample?
Control Types: Is it ever useful to do a non-specific antibody first, then a specific antibody?
Categories: ChIP-Seq Data Analysis
Views: 874 Asked By:Control Types: Is there a bias with ChIP-Seq data because promoters tend to be GC-rich and tend to be open chromatin regions?
Categories: ChIP-Seq Data Analysis
Views: 679 Asked By:Is there a bias with ChIP-Seq data because promoters tend to be GC-rich and tend to be open chromatin regions?
Control Types: Is there a reason not to use both IgG and Input controls?
Categories: ChIP-Seq Data Analysis
Views: 762 Asked By:Control Types: Under what conditions is it better to use the Input versus IgG control for ChIP-Seq?
Categories: ChIP-Seq Data Analysis
Views: 2220 Asked By:Control Types: What is copy number bias? How are we supposed to address it for ChIP-Seq?
Categories: ChIP-Seq Data Analysis
Views: 658 Asked By:Control Types: What is the difference between input and naked DNA?
Categories: ChIP-Seq Data Analysis
Views: 1113 Asked By:What is the difference between in put and naked DNA?
Control Types: Why does my IgG control sample have higher binding than the antibody binding sample for qPCR? What do I do about it?
Categories: ChIP-Seq Data Analysis
Views: 675 Asked By:CSVKit issue in biowulf
Categories: BIOSTAR
Views: 1907 Asked By:Customizing your terminal prompt?
Categories: BIOSTAR
Views: 1727 Asked By:Data visualization example code
Categories: R
Views: 27 Asked By:Difference between 3' and 5' gene expression profiling? Which should I use?
Categories: Single-Cell RNA-Seq
Views: 13046 Asked By:Do I need to have extensive bioinformatic knowledge to explore my Cell Ranger results?
Categories: Single-Cell RNA-Seq
Views: 4592 Asked By:Droplet-based (e.g. 10x Genomics) or plated-based (e.g. Smart-Seq)? When should one be considered over the other
Categories: Single-Cell RNA-Seq
Views: 8812 Asked By:efetch, bgzip, infoseq, seqkit, bioawk – commands not found. Do I need to install something?
Categories: BIOSTAR
Views: 2660 Asked By:Update: found the problem - I had not activated bioinfo in the new terminal window! Its all working now. :)
Leaving this question here in case anybody else is facing the same issue.
Original question - Most of the commands described in the data compression and subsequent chapters are not working - On trying commands like efetch, bgzip, infoseq, seqkit, bioawk etc, I get 'command not found'. Is there any additional the needs to be installed for these?
Elizabeth Introduction
Views: 4318 Asked By:Error in library(DESeq)
Categories: BIOSTAR
Views: 3003 Asked By:I am running the command line shown in chapter XXII (cat simple_counts.txt | Rscript deseq1.r 3x3 > result.txt)
This returned an error message "dyld: Library not loaded: @rpath/libreadline.6.2.dylib
Referenced from: /Users/yangc2/miniconda3/envs/bioinfo/lib/R/lib/libR.dylib
Reason: image not found
Abort trap: 6".
I also tried the other Rscripts deseq2.r and edger.r. But all gave a similar error message and no correct output file. Is there a way to resolve this?
I am running this in Ubuntu/PC.
=============================================
Kudos to Amy's feedback. It turned out to be some setting issue with the r software package. This issue is resolved by re-running the codes as follow:
conda install r
conda install -y bioconductor-deseq bioconductor-deseq2 bioconductor-edger r-gplotsError using parallel tool for fast-dump
Categories: BIOSTAR
Views: 1974 Asked By:cat runids.txt | parallel fastq-dump -X 10000 --split-filesExporting plots with R
Categories: R
Views: 28 Asked By:For single nuclei RNA-Seq, do I have to do anything different in handling the data?
Categories: Single-Cell RNA-Seq
Views: 4524 Asked By:Genetic Variants and ChiP-seq data: Can you use ChIP-seq data to analyze copy number variants? Can you do structural. variant calling?
Categories: ChIP-Seq Data Analysis
Views: 802 Asked By:Genetic Variants and ChIP-seq data: When we are working with patient samples for ChIP-seq, we align them all to one generic reference genome. Will the alignment tolerate and SNPs in the individual patient line?
Categories: ChIP-Seq Data Analysis
Views: 614 Asked By:When we are working with patient samples for ChIP-seq, we align them all to one generic reference genome. Will the alignment tolerate any SNPs in the individual patient line?
Help with R syntax
Categories: R
Views: 29 Asked By:how can i get help in this class?
Views: 4350 Asked By:How can I use a large data set like Tabula Muris for identifying cell types in my data?
Categories: Single-Cell RNA-Seq
Views: 5458 Asked By:How many cells can I capture in my experiment?
Categories: Single-Cell RNA-Seq
Views: 1876 Asked By:How to do the exercise from the book with Chr22 and hg38 but with hg37
Categories: BIOSTAR
Views: 1902 Asked By:I typed this command
$ curl https://hgdownload.soe.ucsc.edu/goldenPath/hg19/chromosomes/chr22.fa.gz | gunzip -c > ~/refs/hg37/chr22.fa
But It just show up the next
% Total % Received % Xferd Average Speed Time Time Time Current
Dload Upload Total Spent Left Speed
0 0 0 0 0 0 0 0 --:--:-- --:--:-- --:--:-- 0
How to enable colors for the ls command
Categories: BIOSTAR
Views: 935 Asked By:How to install Ubuntu if you are located at NCI Frederick
Categories: BIOSTAR
Views: 904 Asked By:How to quickly activate bioinfo in biowulf?
Categories: BIOSTAR
Views: 1792 Asked By:[user@biowulf temp]$ source /data/$USER/conda/etc/profile.d/conda.sh [user@biowulf temp]$ conda activate bioinfo
I tried both creating a bioinfo.sh shell script and defining an alias called bioinfo to
quickly activate bioinfo, but all failed. Could you please help me with that? Thanks
how to use commend lines to do data analysis?
Categories: BIOSTAR
Views: 1596 Asked By:- I just copy from biostar and paste them into terminal and ran well with those commends according to biostar book But I still do not know how to do them by myself. Do you guys have any good suggestions about what I should do to learn the knowledge from the book to use for my own use? instead of copy and paste from biostar website?
- Could you help to demonstrate how to do data analysis with unix commend lines in terminal in the class, instead of just follow the book to copy and paste the commends lines into terminal? That will be more helpful and help us to learn how to use the commend to solve the problems.
I had great results with ChIP-qPCR, but when I did ChIP-seq only a few reactions came up with decent peaks. What could be the reason?
Categories: ChIP-Seq Data Analysis
Views: 1902 Asked By:Introduction
Views: 4679 Asked By:Introduction
Views: 4233 Asked By:Hi everyone! My name is Mythili Merchant and I am a postbac in Dr. Jing Wu's lab at the Neuro-Oncology Branch in Bethesda! My projects are mainly focused on understanding the landscape of gliomas and development of therapeutics.
I have enjoyed bioinformatics for a long time, but have always focused on using GUI for database mining mainly to characterize functions of hypothetical genes. However, I have been hoping to learn more about working with NGS data and to learn how to analyze and use it. Also, hoping to pick up some other useful skills along the way!
Looking forward to getting to know everyone! See you all on Thursday at 3 pm.
Introduction - Corey Young
Views: 4514 Asked By:Issues with installing jellyfish on a MAC computer
Categories: BIOSTAR
Views: 1869 Asked By:I am trying to install jellyfish on my Mac computer. Typing "make install" gives me an error like:
/usr/local/lib/libjellyfish-2.0.2.dylib: Permission denied
or
mkdir: /usr/local/lib: Permission denied
Joanne Introduction
Views: 4139 Asked By:Labeling select data points on a plot
Categories: R
Views: 40 Asked By:Lecture recording and notes
Categories: BIOSTAR
Views: 1854 Asked By:Are our lectures/classes recorded, and if so where can we access them?
Library ChIP preparation: Can ChIP-seq be done on formalin fixed tissue and if yes how much starting material is usually required?
Categories: ChIP-Seq Data Analysis
Views: 788 Asked By:Library ChIP preparation: How do I choose between sonication and enzymatic-based chromatin fragmentation for ChIP-seq? How might this affect sequencing depth?
Categories: ChIP-Seq Data Analysis
Views: 1006 Asked By:How do I choose between sonication and enzymatic-based chromatin fragmentation for ChIP-seq? How might this affect sequencing depth?
Library ChiP preparation: Is the sequencing depth based on the organism you're working with?
Categories: ChIP-Seq Data Analysis
Views: 751 Asked By:Library ChIP preparation: What happens if there are too many PCR cycles?
Categories: ChIP-Seq Data Analysis
Views: 879 Asked By:Library ChIP preparation: What should be the starting amount of DNA for fresh and fixed sample?
Categories: ChIP-Seq Data Analysis
Views: 737 Asked By:Library/ChIP preparation: Is it okay to use frozen samples or is it better to use fresh cells?
Categories: ChIP-Seq Data Analysis
Views: 897 Asked By:Library/ChIP preparation: What are nanobodies? Can they be used for ChIP-Seq?
Categories: ChIP-Seq Data Analysis
Views: 2071 Asked By:What are nanobodies? Can they be used for ChIP-Seq?
Library/ChIP preparation: What if my antibody has low binding affinity and there isn't a better option available?
Categories: ChIP-Seq Data Analysis
Views: 720 Asked By:Local vs. global alignment
Categories: BIOSTAR
Views: 5688 Asked By:MacOS users: Where to find older versions of XCode?
Categories: BIOSTAR
Views: 22835 Asked By:In going through the macOS computer setup instructions in the biostar handbook, the XCode version in the App Store requires macOS version 10.15.2 or later. What if I am using an older version of the macOS?
Makefile target command problems
Categories: BIOSTAR
Views: 1124 Asked By:More trouble with Xcode
Categories: BIOSTAR
Views: 1180 Asked By:Peak calling and analyses: If the control is input, which method peak calling do you prefer?
Categories: ChIP-Seq Data Analysis
Views: 1979 Asked By:Peak calling and analyses: What do you think about ChIPseeker for annotation?
Categories: ChIP-Seq Data Analysis
Views: 2170 Asked By:Peak calling and analyses: What peak caller is good for broad peaks?
Categories: ChIP-Seq Data Analysis
Views: 2081 Asked By:Peak calling and analyses: When I call peaks with MACS2, can I use one ChIP (antibody1) as the control for a second ChIP antibody? Can I detect differences between the two proteins that way?
Categories: ChIP-Seq Data Analysis
Views: 2742 Asked By:When I call peaks with MACS2, can I use one ChIP (antibody1) as the control for a second ChIP antibody? Can I detect differences between the two proteins that way?
Pipelines and QC: Does the mouse genome have a blacklist file?
Categories: ChIP-Seq Data Analysis
Views: 2083 Asked By:Pipelines and QC: Since reads are mapped to unique regions of the genome, what happens with data from repeat regions such as long non-coding RNA, LINEs, etc? Will any reads be mapped to these regions?
Categories: ChIP-Seq Data Analysis
Views: 741 Asked By:Pipelines and QC: What are "no hits" on a multiQC report? Are they all adaptors?
Categories: ChIP-Seq Data Analysis
Views: 778 Asked By:Pipelines and QC: What is "effective genome size"?
Categories: ChIP-Seq Data Analysis
Views: 2032 Asked By:Pipelines and QC: What is a good mapping percentage?
Categories: ChIP-Seq Data Analysis
Views: 863 Asked By:Pipelines and QC: What is the average length of a read produced by ChIP-seq?
Categories: ChIP-Seq Data Analysis
Views: 700 Asked By:Pipelines and QC: When and how do you remove blacklisted regions? What program do you use?
Categories: ChIP-Seq Data Analysis
Views: 3482 Asked By:Pipelines and QC: Which ChIP-seq pipelines mentioned in your talk can handle paired-end reads?
Categories: ChIP-Seq Data Analysis
Views: 767 Asked By:Pipelines and QC:What package do you use to perform a partial duplicate removal?
Categories: ChIP-Seq Data Analysis
Views: 1950 Asked By:Practicing R with DNAnexus
Categories: R
Views: 27 Asked By:questions related to single cell ATAC-seq?
Categories: Single-Cell RNA-Seq
Views: 2142 Asked By:Replicates: Can you talk a little bit about IDR? Is IDR something you must do for transcription factor ChiP-seq or is it optional?> What about with histone marks? Does IDR work with MACS2 outputs?
Categories: ChIP-Seq Data Analysis
Views: 2688 Asked By:Replicates: If my replicates are highly reproducible, is it valid to merge my replicates before finding peaks?
Categories: ChIP-Seq Data Analysis
Views: 2789 Asked By:Ryan Introduction
Views: 4260 Asked By:Should we always use histone markers just to see if our sequencing is working for our samples?
Categories: ChIP-Seq Data Analysis
Views: 1990 Asked By:Spike-ins: At what stage of the ChIP experiment do you introduce the spike-ins?
Categories: ChIP-Seq Data Analysis
Views: 763 Asked By:Spike-ins: Can you please give me more detail on how the spike-in works?
Categories: ChIP-Seq Data Analysis
Views: 986 Asked By:Spike-ins: How much spike-in is a good amount to use?
Categories: ChIP-Seq Data Analysis
Views: 823 Asked By:How much spike-in is a good amount to use?
Spike-ins: What is a good spike-in sample for ChIP-seq?
Categories: ChIP-Seq Data Analysis
Views: 907 Asked By:The miniconda download does not work!
Categories: BIOSTAR
Views: 1378 Asked By:It does not download, I just get an error message.
Trouble building CTAT custom genome
Views: 4609 Asked By:trouble installing makefiles
Categories: BIOSTAR
Views: 1598 Asked By:I used conda install make, but the condo install makefiles does not work. I get this error:
$ conda install Makefiles
Collecting package metadata (current_repodata.json): done
Solving environment: failed with initial frozen solve. Retrying with flexible solve.
Collecting package metadata (repodata.json): done
Solving environment: failed with initial frozen solve. Retrying with flexible solve.
PackagesNotFoundError: The following packages are not available from current channels:
- makefiles
Current channels:
- https://conda.anaconda.org/bioconda/osx-64
- https://conda.anaconda.org/bioconda/noarch
- https://conda.anaconda.org/conda-forge/osx-64
- https://conda.anaconda.org/conda-forge/noarch
- https://repo.anaconda.com/pkgs/main/osx-64
- https://repo.anaconda.com/pkgs/main/noarch
- https://repo.anaconda.com/pkgs/r/osx-64
- https://repo.anaconda.com/pkgs/r/noarch
To search for alternate channels that may provide the conda package you're
looking for, navigate to
https://anaconda.org
and use the search bar at the top of the page.
(bioinfo)
Ubuntu Linux on Windows link
Categories: BIOSTAR
Views: 1176 Asked By:Ubuntu on Windows installation issues
Categories: BIOSTAR
Views: 1631 Asked By:Unix Quiz 1
Views: 4298 Asked By:What are the Rules and Purpose of this Forum
Categories: BIOSTAR
Views: 5097 Asked By:What are the Rules and Purpose of this Forum
Views: 1699 Asked By:What do you think about ChIP-seq with proteins that are highly mobile in nature? Why is there less data published about using them?
Categories: ChIP-Seq Data Analysis
Views: 1875 Asked By:what does -f stand for in the bash profile setup?
Categories: BIOSTAR
Views: 3621 Asked By:# if [ -f ~/.bashrc ]; then source ~/.bashrc fi
What is Cell Ranger?
Categories: Single-Cell RNA-Seq
Views: 4624 Asked By:What is the difference between RPKM, FPKM and TPM
Categories: FAQ
Views: 70858 Asked By:What sequencing depth do I need for scRNA-Seq samples?
Categories: Single-Cell RNA-Seq
Views: 6355 Asked By:When should we batch correct scRNA-Seq data? When should we avoid it?
Categories: Single-Cell RNA-Seq
Views: 17015 Asked By:Which cell types can be used in Single-Cell RNA Seq?
Categories: Single-Cell RNA-Seq
Views: 1405 Asked By:Which shell to use for Mac OS Catalina (10.15.*)
Categories: BIOSTAR
Views: 1330 Asked By:Why don’t the scRNA transcript counts match up with the results I found with flow cytometry?
Categories: Single-Cell RNA-Seq
Views: 4995 Asked By:Will we cover R/Python for bioinformatics in this course?
Views: 5008 Asked By: