What factors should I consider when switching from QuantSeq-FWD to QuantSeq-Pool?
When switching from QuantSeq-FWD to QuantSeq-Pool, there are certain considerations researchers should take to ensure QuantSeq-Pool is the appropriate kit for their application.
General comparison between QuantSeq-FWD (Cat. No. 191-196) and QuantSeq-Pool (Cat. No. 139)
QuantSeq-FWD | QuantSeq-Pool | |
|---|---|---|
Input amounts | 1ng - 500ng* *low input protocol modifications available for 1 ng - 10 ng. | 10ng - 120ng per sample into First Strand Synthesis. 80ng - 960ng per pool into Second Strand Synthesis. |
RNA quality | High and low quality RNA is acceptable, as well as FFPE RNA. | Medium to high quality RNA required (i.e., RIN >6). |
Blood samples | Compatible with Globin Block Modules | Not recommended. |
Unique Molecular Identifiers (UMIs) | Optional with UMI Second Strand Synthesis Module (Cat. No. 081). | Included. |
First Strand Synthesis Primer | Oligo(dT) primer with partial Illumina adapter sequence. | Oligo(dT) primer with 12 nt i1 barcode, 10 nt UMI, and partial Illumina adapter sequence. |
Library Quality Control (QC) | QC individual libraries and pool at equimolar ratio. | QC final, pooled library; individual library QC not possible due to early pooling after reverse transcription. |
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. |
Sequencing mode | Single Read (SR) Read 1: insert; 50 – 150bp | Paired End Read (PE) |
Demultiplexing | i5/i7 demultiplexing required.
| i5/i7 demultiplexing optional (if UDI are used). i1 demultiplexing required (iDemux recommended). If only using Lexogen 12 nt UDI in your sequencing lane (e.g., with other Lexogen libraries), we recommend iDemux for demultiplexing and optimal error correction. |
What components are interchangeable between QuantSeq-FWD and QuantSeq-Pool?
FS in QuantSeq-Pool and FS1/FS2 in QuantSeq-FWD | Not interchangeable |
First Strand Synthesis Enzyme (E1) | Interchangeable |
Purification Module (PB, PS, EB) | Interchangeable |
RPM in QuantSeq-Pool and SS1/SS2 in QuantSeq-FWD | Not interchangeable |
Second Strand Synthesis Enzyme (E2) | Not interchangeable |
Library Amplification Module | PM and PE are interchangeable NOTE: P5 and P7 required for QuantSeq-Pool when no i5/i7 indexing is done. |
When switching from QuantSeq-FWD to QuantSeq-Pool, we strongly encourage you to perform a pilot experiment, to ensure you are satisfied with the data. If performing a head-to-head comparison of QuantSeq-FWD and QuantSeq-Pool, consider the following:
Use the same sample type and same input amounts.
Include technical replicates to assess handling variability (QuantSeq-FWD samples are processed separately whereas QuantSeq-Pool samples are processed together).
Optional: Include spike-in RNA controls (SIRVs) for assessing differential gene expression to determine “ground truth” (comparing expected vs observed).
Compare samples using the same read depth and downsample if necessary (e.g., if you have 10 million reads for Sample A and 1 million reads for Sample B, randomly discard a specified fraction of reads so Sample A and Sample B both have 1 million reads.). If you have questions about downsampling, please contact support@lexogen.com.
Use the same Read 1 length (i.e., insert length). We recommend >75bp for Read 1.
Do not collapse UMIs, even if UMIs are included in QuantSeq-FWD. It is instead advised to check the correlation of non-collapsed samples.