Do you have recommended loading concentrations for Lexogen libraries on Illumina sequencing instruments?
Below you can find a table of advised loading concentrations for different Lexogen library pools on common Illumina sequencing platforms. If specified ranges are provided these are based either on Lexogen’s own validation of loading concentrations, and/or feedback provided by Lexogen customers, and are known to contribute to optimal sequencing quality and output. These recommendations apply to pure lanemixes per library type. If you are running lanemixes featuring a mixture of different library prep types, loading concentrations may need to be optimized specifically.
PhiX spike-in recommendations are also provided. Please note PhiX should not be added to QuantSeq REV runs. Due to the use of a custom sequencing primer (CSP) PhiX will not be detected.
NOTE: Where no specific loading concentration / PhiX details are provided please refer to the Illumina loading concentration recommendations for the specific instrument and/or flow cell. We can generally recommend starting at the higher end of these Illumina loading concentration ranges.
QuantSeq FWD (Cat. No.s 191-196) & QuantSeq-Flex (Cat. No.s 166, 028) | QuantSeq FWD with UMIs (Cat. No.s 191-196 + 081) | QuantSeq-Pool (Cat. No. 139) | QuantSeq REV* (Cat. No. 225) | CORALL (Cat.no.s 171-176, 177-182, 183-186) | LUTHOR HD (Cat. No.s 204, 221) | Small RNA-Seq (Cat. No.s 052, 058) | |
|---|---|---|---|---|---|---|---|
NextSeq 500 / 550 2 | 2 - 2.5 pM4 (+ 1 - 5% PhiX) | 1.8 - 2 pM4 (+ 15 - 30% PhiX) 1 | 2 - 2.5 pM4 (+ 1 - 5% PhiX) | 2 - 3 pM4 | 2.2 - 2.7 pM4 (+ 1 - 5% PhiX) | 2 - 2.5 pM3 (+ 1 - 5% PhiX) | 2.6 pM4 (5 % PhiX) |
NextSeq 2000 3 | 650 - 750 pM (+ 1 - 5% PhiX) | 750 pM (+ 30% PhiX) 1 | 700 - 800 pM (+ 1 - 5% PhiX) | ** | 650 - 750 pM (+ 1 - 5% PhiX) | ** | 650 pM |
MiSeq | 6 – 15 pM (+ 1 - 5% PhiX) | 11 pM (+ 5% PhiX) 1 | ** | 6 – 15 pM | ** | 6 - 15 pM (+ 1 - 5% PhiX) | 15 – 20 pM |
MiniSeq | 1.3 – 1.8 pM (+ 1 - 5% PhiX) | ** (+ 15 - 30% PhiX) 1 | ** | ** | ** | 1.3 - 1.8 pM (+ 1 - 5% PhiX) | ** |
NovaSeq 6000, X, X Plus | 200 - 400 pM (+ 1 - 5% PhiX) | ** (+ 15 - 30% PhiX) 1 | ** | ** | ** | Standard: 300 - 500 pM, | ** |
iSeq 100 | ** | ** (+ 15 - 30% PhiX) 1 | ** | Not compatible!5 | ** | ** | ** |
* PhiX should not be added to REV runs. Due to the use of a custom sequencing primer (CSP), it will not be detected.
** No specific guidelines available. Please follow the advised loading concentration ranges for the specific instrument, as specified by Illumina - or contact support@lexogen.com.
1 Higher PhiX spike-in is required to overcome a low diversity spacer sequence located after the UMI at positions 7 - 10 of read 1. The low diversity stretch can lead to reduced run output by negatively impacting Q30 values and % PF rate for the run. In most cases 15% PhiX will be sufficient to offset these impacts.
2 The loading concentrations provided for NextSeq 500 have been evaluated by Lexogen to produce optimal cluster densities of 200 - 260 K/mm2. Loading concentrations below 2 pM can result in lower cluster densities and reduced index read quality, leading to reduced run output.
3 Loading concentrations are still under evaluation by Lexogen for the NextSeq 2000 platform and will be updated periodically.
4 This loading amount aims for a cluster density of 250 K/mm2 on the NextSeq 500, as optimized by the Lexogen Services Facility. Please note optimal cluster density vs. run performance may vary on individual instruments and should best be specifically optimized.
5 The iSeq 100 instrument does not support the use of Custom Sequencing Primers.