General comparison between QS (Cat. No. 191-196) and QSP (Cat. No. 139):
QuantSeq-FWD | QuantSeq-Pool | |
|---|---|---|
Input amount | 1ng - 500ng* *lower inputs possible with protocol modifications | 10ng - 120ng per sample into FSS 80ng - 960ng per pool into SSS |
RNA quality | N/A - both high and low quality RNA is acceptable, as well as FFPE RNA | Medium-high quality RNA (RIN >6) |
First Strand Synthesis Primer | Oligo(dT) primer with partial Illumina adapter sequence | Oligo(dT) primer with 12nt i1 barcode, 10nt UMI, and partial Illumina adapter sequence |
Library QC | QC individual libraries and pool at equimolar ratio | QC pooled library; individual library QC not possible |
Average Library Size | ~335 – 456 bp (based on UHRR)* *Can vary based on RNA quality and sample type | ~650 bp (based on UHRR)* *Can vary based on RNA quality and sample type |
Demultiplexing | i5/i7 demultiplexing required (can be done with bcl2fastq) | i5/i7 demultiplexing optional i1 demultiplexing required |
What components are interchangeable:
FS in QSP and FS1/FS2 in QSF | Not interchangeable |
FSS enzyme (E1) | Interchangeable |
Purification module | Interchangeable |
RPM in QSP and SS1/SS2 in QSF | Not interchangeable |
SSS enzyme (E2) | Not interchangeable |
Library Amplification Module | PM and PE are interchangeable P5 and P7 required for QSP when no i5/i7 indexing is done |
If performing a head-to-head comparison of QuantSeq-FWD and QuantSeq-Pool, consider the experimental set-up:
Use same input amount
Compare samples at same read depth - down sample if necessary
Same Read 1 length
Do not collapse UMIs, even if UMIs are included in QuantSeq-FWD (with SSS UMI Module)
Recommend including technical replicates to assess handling variability (QSF samples processed separately whereas QSP samples processed together)
Recommend including controls (SIRVs) to assess DEG, if interested in this (compare expected vs observed)
What can also cause differences:
Differences in chemistries and protocol