Page Comparison

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

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QuantSeq-FWD (Cat. No. 191-196) and

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QuantSeq-Pool (Cat. No. 139)

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	QuantSeq-FWD	QuantSeq-Pool
Input

amount

amounts

1ng - 500ng* *

lower inputs possible with

low input protocol modifications available for 1 ng - 10 ng.

10ng - 120ng per sample into

FSS

First Strand Synthesis. 80ng - 960ng per pool into

SSS

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 Globin Block Modules (Cat. No. 070 (Homo sapiens) and 071 (Sus scrofa)).	Not recommended.
Unique Molecular Identifiers (UMIs)	Optional with UMI Second Strand Synthesis Module (Cat. No. 081). UMIs are 6 nt long (plus a 4 nt spacer) and added during Second Strand Synthesis.	Included. UMIs are 10 nt long 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 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) Read 1: insert; 50 – 150bp Read 2: UMI and i1 barcode; 22bp
Demultiplexing	i5/i7 demultiplexing required

(can be done with bcl2fastq)

. 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.

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

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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 if necessarySame Read 1 lengthand 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 (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). It is instead advised to check the correlation of non-collapsed samples.