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changes.mady.by.user Lauren Sheehan

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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
I prefer not to mention any input lower than 1 ng.
I would rather indicate:
1 ng - 500 ng*
*low input protocol modification for 1 ng - 10 ng

10ng - 120ng per sample (first strand synthesis or FSS) into FSS

80ng - 960ng per pool (second strand synthesis or SSS) 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)

Blood samples

Compatible with RS-Globin block modules
cat n° 070 (Homo sapiens)
cat n° 071 (Sus scrofa)

Not recommended

UMI

optional with UMI Add-on Cat n° 081.
UMI are 6 nt long (with a 4-nt spacer) and added during second strand synthesis

included
10 nt long
Added during first strand synthesis

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

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 for optimal error correction

i1 demultiplexing required (idemux recommended)

i5/i7 demultiplexing optional

i1 demultiplexing required

If only using Lexogen 12 nt UDI in your sequencing lane, we recommend idemux for optimal error correction

What components are interchangeable:

FS in QSP and FS1/FS2 in QSF

Not interchangeable

FSS enzyme (E1)

Interchangeable

Purification module (PB, PS, EB)

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

Note

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 (e.g., if you have 10 million reads in library A and 1 million reads in library B, you need to randomly - through bioinformatics - decrease the number of analysed reads in library A down to 1 million)

  • Same Read 1 length (i.e., insert length)

  • 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:

...