Page Comparison

General comparison between QS 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 amountamounts	1ng - 500ng* * lower inputs possible with protocol modifications I prefer not to mention any input lower than 1 ng. I would rather indicate: 1 ng - 500 ng* * low input protocol modification modifications available for 1 ng - 10 ng.	10ng - 120ng per sample (first strand synthesis or FSS) into FSSinto First Strand Synthesis. 80ng - 960ng per pool (second strand synthesis or SSS) into SSSinto Second Strand Synthesis.
RNA quality	N/A - both high 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 RS- Globin block modules cat n° Block Modules (Cat. No. 070 (Homo sapiens) cat n° and 071 (Sus scrofa)).	Not recommended	UMI	optional with UMI Add-on Cat n° 081. UMI .
Unique Molecular Identifiers (UMIs)	Optional with UMI Second Strand Synthesis Module (Cat. No. 081). UMIs are 6 nt long ( with plus a 4 - nt spacer) and added during second strand synthesis included Second Strand Synthesis.	Included. UMIs are 10 nt long Added during first strand synthesis and added during First Strand Synthesis.
First Strand Synthesis Primer	Oligo(dT) primer with partial Illumina adapter sequence.	Oligo(dT) primer with 12nt 12 nt i1 barcode, 10nt 10 nt UMI, and partial Illumina adapter sequence.
Library Quality Control (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.
Sequencing mode	Single Read (SR)	Paired End Read (PE) Read 1: insert Read 2: UMI and i1 barcode
Demultiplexing	i5/i7 demultiplexing required (can be done with bcl2fastq) I would like to promote idemux here . Suggestion: If only using Lexogen 12 nt UDI in your sequencing lane, we recommend idemux iDemux for demultiplexing and optimal error correction.	i5/i7 demultiplexing optional. i1 demultiplexing required ( idemux iDemux recommended). i5/i7 demultiplexing optional If only using Lexogen 12 nt UDI in your sequencing lane, we recommend idemux iDemux for demultiplexing and optimal error correction.

What components are interchangeable

...

between QuantSeq-FWD and QuantSeq-Pool?

• Use the same input amountsample 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 at using the same read depth - down sample and downsample if necessary (e.g., if you have 10 million reads in library for Sample A and 1 million reads in library for Sample B, you need to randomly - through bioinformatics - decrease the number of analysed reads in library A down to 1 million)Same 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 (with SSS UMI Module)

• Include Recommend including technical replicates to assess handling variability (QSF samples processed separately whereas QSP samples processed together)

• Include Recommend including controls (SIRVs) as “ground truth” (compare expected vs observed) to assess differential gene expression DEG, if interested in this parameter (compare expected vs observed)

What can also cause differences:

• Differences in chemistries and protocol. It is instead advised to check the correlation of non-collapsed samples.