Question: What are alternative sequencing formats for Single Cell V(D)J libraries?
Answer: For B- and T-cell Enriched V(D)J Libraries, we recommend the following sequencing configuration:
- Read 1: 150 cycles, i7 Index: 8 cycles, Read 2: 150 cycles
The i7 index read must not be shorter than recommended. Application performance may be affected if Read 1 and/or Read 2 are shorter or longer.
Sequencing less than 2 x 150 bp may reduce application performance. Specifically, Read 2 length is critical for spanning V-J junctions. In silico testing of read lengths shorter than 150 bp suggests that 2 x 100 bp sequencing can reduce performance metrics (total number of detected cells with productive V-J spanning TRA/TRB or IGH and IGK/IGL pairs). Furthermore, 2 x 75 bp sequencing will not result in usable data as the TCR or Ig transcripts will not be assembled.
Sequencing longer than 2 x 150 bp may lead to reduced sequence accuracy where the low Q30 ends of the two reads meet, particularly on the longer V(D)J transcripts.
For 5' Gene Expression Libraries, we recommend the following sequencing configuration:
- Read 1: 26 cycles, i7 Index: 8 cycles, Read 2: 98 cycles
Read 1 and i7 index reads must not be shorter than indicated. Read 2 can be lengthened or shortened but the altered length may affect transcript mapping rates.
For more information, see our Technical Note on Base Composition of Sequencing Reads of Chromium Single Cell VDJ Libraries (https://support.10xgenomics.com/single-cell-vdj/index/doc/technical-note-base-composition-of-sequencing-reads-of-chromium-single-cell-vdj-libraries)