Web summary metrics for V(D)J Troubleshooting

The number of barcodes is estimated to be associated with cells that express targeted V(D)J transcripts. Depends on the expected number of T/B cells.

Lower or higher than expected V(D)J cell calling could be due to poor sample quality, poor library quality, or poor sequencing quality.

• If cells are overcalled, cellranger multi is recommended to remove the background*.
• If cells are undercalled, consider looking at Reads mapped to any V(D)J genes (below), and contacting 10x Genomics Technical Support with library enrichment traces.

Fraction of reads that partially or wholly map to any germline V(D)J gene segment.

• Ideal > 50% ; > 40% is an acceptable limit.

This is likely related to the proportion of B or T cells present in the sample.  Samples with a low value may have a limited number of B or T cells.  Enrichment may be needed for these samples, and please review the recommended methods highlighted in this KB article. 

In a few other cases, this could be caused due to wrong chemistry or wrong reference used.

Fraction of cell-associated barcodes with at least one productive contig for each chain of the receptor pair.

• Ideal > 30% ; >20% is an acceptable limit
  

Lower values could indicate poor sample quality, poor or low enrichment, poor coverage, or low sequencing depth. There may be a few reasons why a single chain may be captured or expressed and it is explained in this article. In rare cases, wrong chemistry could also give rise to a low value.

The median number of UMIs assigned to a {chain} contig per cell (where a chain is TRA/TRB or IGH/IGK/IGL). 

• Ideal >0 UMIs 

Higher median UMI counts indicate higher support for a chain expression. 0 median UMIs often indicate poor expression of TCR/BCR chains, poor sample quality, or poor/low enrichment in some cases. In rare cases, missing chains may be reflective of the biology of the samples.

TRA transcripts are often expressed at lower levels than TRB transcripts.  Please review this KB article for additional information.

If GEX data is available, Loupe files can be used to identify the expression of T/B- cells using the steps illustrated here.

Based on the experiment, assess the plot for what fraction of observed barcodes are called cells and the range of UMI counts per barcode. 

The count of filtered UMIs mapped to each barcode on X-axis is provided on the Y-axis. The color of the graph is based on the local density of cell-associated barcodes. 

Valid Barcodes

Fraction of reads with barcodes that match the whitelist after barcode correction.

• Ideal > 85%
• Acceptable is >75%

Low valid barcode warning, can occur for several reasons:

• It is often related to poor sample quality, sequencing quality, or improper sequencing parameters. 
• In rare cases, this warning may be due to the incomplete removal of ethanol wash solution during SPRI cleanups which may result in the amplification of undesired products, which can sometimes result in lower valid barcodes. Please review this KB article.

The histogram plot displays the fraction of cells occupied by the 10 most abundant clonotypes in any sample. In good samples, most of these clonotypes will have both chains.

If a large clonotype fraction with multiple chains is noticed, it is highly suggestive of ambient RNA*. Cell Ranger v5 and above implements a better clonotype grouping algorithm. The presence of an identical chain in multiple clonotypes is also an indication of ambient RNA.