mshadbolt
commented
5 years ago Overall understanding of 10X VDJ sequencing so far
Input: dissociated cells as cell suspensions
Output: >=2 library preparations from a single cell suspension.
The cell barcoded cDNA from the cells in the cell suspension is split between the library preparations after amplification. 1 library is standard 10X 5' style gene expression (gex). The other libraries are the result of a PCR enrichment step using primers. There is an Enrichment kit that can be used to enrich for either B cells or T cells. You can enrich for both from the same cell suspension input. This would result in 3 different libraries, 5 prime tag based expression, paired end T cell VDJ sequences, paired end B cell VDJ sequences. The VDJ sequences may either have the same read lengths as the gex libraries or one can do paired end 150bp reads. The cell barcoding and UMI layout is the same and occur at the start of read 1.
Reads from each VDJ enrichment library are assembled or aligned to known VDJ sequences.
The current dataset that I am wrangling (Kylie James Colon Immune cells) has the three libraries as described above.
How to fill relevant metadata fields
Library preparation protocol
Should there be a single library prep protocol for all libraries or a separate one for each library type?
The way I have modelled it in my experiment is to have separate protocols for each library, e.g.
10x_v2_5p_gex_library_prep_protocol
10x_v2_5p_vdj_TCR_library_prep_protocol
10x_v2_5p_vdj_Ig_library_prep_protocol
This would enable the user to see which sequence file derived from each library. Open to suggestions if this is a good idea or not.
Potential new metadata fields
Should we add a field to the library_preparation_protocol module that would capture the 'enrichment' primers?
Does the analysis team need any specific fields added that would need to be captured to enable analysis? (Perhaps too early to tell if pipelines for this data are a long way off)
Potential new Ontology terms
To populate library_preparation_protocol.library_construction_method.ontology we will need a new V(D)J specific term
Should the term be something like 10X 5' v2 V(D)J sequencing?
Should this sit underneath 10X 5' v2 sequencing ?
Should the gex libraries also get this term or only the vdj specific libraries?
It seems like so far there are v1 and v1.1 versions of V(D)J libraries, but I'm not sure the difference so not sure if we need to have both ontology terms.
To populate library_preparation_protocol.input_nucleic_acid_molecule.ontology should we request a more specific term to indicate polyA RNA from TCR (T cells) or Ig (B cells) ?
References:
Chromium Single Cell V(D)J Reagent Kits - User Guide 10x-pert Workshop | Characterization of the Tumor Microenvironment with the Chromium Single Cell Imm Sequencing Requirements for Single Cell V(D)J Experimental design for V(D)J libraries Chromium VDJ presentation with nice diagrams
lauraclarke
ESapenaVentura
clairerye
Description
As a data wrangler wrangling a project with 10X 5' V(d)J data I need to determine the correct way of defining the experiment in our spreadsheet and determine whether any additional fields or ontology terms are needed. Firstly I will need to get a better understanding of what V(D)J sequencing is and how it differs from 10X 5' sequencing. I heard that perhaps @ambrosejcarr and perhaps @TimothyTickle had started thinking about this so please provide any opinions if you have any. Not sure where this lies on the analysis pipelines roadmap and what type of information would need to be captured above and beyond what we would capture from a standard 10X 5' sequencing library **Acceptance Criteria**e.g.: