Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • Deal with RNA spike-in in single cell analysis

    Hi everyone,

    I'm working with single cell RNAsequencing data obtained with the fluidigm technology. In the protocol, we chose the 96 well-plate which allows us to get full length mRNAseq from the cell.
    We followed the protocol from fluidigm and It doesn't provide ERCC spike-in to normalize the data but Ambion AM1780 spike to control the efficiency of the reaction in the C1. So we add 3 spikes in reaction.

    We received the data from the sequencing and I'm starting to analyze them. I'm a little bit stuck on how to deal with the spike.

    I have a quiescent population of cells, which do not express a lot of gene (2000 detected genes), and the percentage of Ambion spike in these libraries is closed to 50%.
    In an other population of cells, they express close to 8000 gènes, and the representativity of spike are close to 5-10%.

    I started to analyzed the data with Seurat, and in the function Seurat::NormalizeData(), it takes the size of the library to make the normalization.

    As the representativity of the spike is not the same in all the library, I obtain really different results depending on the fact that I include the spike or not in the beginning of the analyses.

    Does anybody get the same problem with this technologie, and know If I need to include the spike-in in the count-table to start the downstream analyses??

    Thanks a lot,
    Nicolas

Latest Articles

Collapse

  • seqadmin
    Exploring the Dynamics of the Tumor Microenvironment
    by seqadmin




    The complexity of cancer is clearly demonstrated in the diverse ecosystem of the tumor microenvironment (TME). The TME is made up of numerous cell types and its development begins with the changes that happen during oncogenesis. “Genomic mutations, copy number changes, epigenetic alterations, and alternative gene expression occur to varying degrees within the affected tumor cells,” explained Andrea O’Hara, Ph.D., Strategic Technical Specialist at Azenta. “As...
    07-08-2024, 03:19 PM
  • seqadmin
    Exploring Human Diversity Through Large-Scale Omics
    by seqadmin


    In 2003, researchers from the Human Genome Project (HGP) announced the most comprehensive genome to date1. Although the genome wasn’t fully completed until nearly 20 years later2, numerous large-scale projects, such as the International HapMap Project and 1000 Genomes Project, continued the HGP's work, capturing extensive variation and genomic diversity within humans. Recently, newer initiatives have significantly increased in scale and expanded beyond genomics, offering a more detailed...
    06-25-2024, 06:43 AM

ad_right_rmr

Collapse

News

Collapse

Topics Statistics Last Post
Started by seqadmin, 07-10-2024, 07:30 AM
0 responses
30 views
0 likes
Last Post seqadmin  
Started by seqadmin, 07-03-2024, 09:45 AM
0 responses
201 views
0 likes
Last Post seqadmin  
Started by seqadmin, 07-03-2024, 08:54 AM
0 responses
212 views
0 likes
Last Post seqadmin  
Started by seqadmin, 07-02-2024, 03:00 PM
0 responses
194 views
0 likes
Last Post seqadmin  
Working...
X