AVITI Loading Guidelines- CloudBreak Freestyle 2025

The loading guidelines provided below are for Lexogen libraries prepared using the Cloudbreak Freestyle workflows for sequencing on the Element Bioscience AVITI sequencing platform. These guidelines are based on the current experience of Lexogen. These guidelines are subject to change in the event of chemistry or AVITI Operating system (AOS) version updates made by Element Biosciences.

Loading amounts should be adjusted depending on average library sizes and in accordance with the recommendations provided by Element Biosciences for the version of the AOS in use. Please consult the applicable CouldBreak Sequencing User Guide (MA-00058) when planning your run.

When sequencing Lexogen libraries for the first time on the AVITI, we recommend using the Individually Accessible Lanes (IAL) feature, and testing 2 different concentrations in 0.5 pM increments, starting at the average, or higher end of the provided loading ranges.

If you need to use the Adept workflow or UltraQ chemistry, or would like help to optimize your run conditions for specific lanemixes, please contact technical support at support@lexogen.com.

When contacting Lexogen technical support, please provide: a copy of the linear lanemix size distribution (trace file) showing average library/lanemix size, which library type(s) are included in the lanemix(es), the AVITI OS version, and sequencing kit to be used.

Cloudbreak Freestyle

To determine loading concentration ranges, Lexogen libraries are classified as “Third Party, PCR-Plus”. Unless otherwise indicated below, 2% PhiX can be used as a routine spike-in. The table below provides both tested and 👉 suggested loading concentration ranges, based on in-house and customer experience. Relevant AOS versions are indicated by superscript numbering.

NOTE: Loading concentrations for AVITI24 instruments follow the recommendations for AOS v3.4 and higher.

*The 2x300 cycle kits are not recommended for sequencing Lexogen RNA-Seq library types.

¹ Although not recommended, QuantSeq FWD libraries can be sequenced in Paired End (PE i.e. PE75-100) mode, if required. In such cases, only Read 1 should be used for downstream QuantSeq data analysis. Note that you will see a quality drop for read 2, due to reading through the polyT-stretch at the start of the read. This may lead to a lower % Pass Filter (%PF) rate. See this FAQ for more information.

² CORALL RTM and CORALL FFPE Libraries can also be sequenced using PE150 read formats. This does not impact %>Q30 or %PF. For QuantSeq FWD/REV/Pool and Luthor HD/HD-Pool libraries, increasing the sequencing read length up to 150 bases (or the maximum allowed cycles when using a 2x75 kit and 12-24 cycle R2 lengths) is also possible. However, per base sequencing quality will typically decrease as read length increases, as more inserts will be fully covered and sequencing starts to read into the poly(A) stretch and adapter sequences.

³Provided run formats correspond to Lexogen’s recommended sequencing formats for each library type. Read 2 sequencing formats for QuantSeq-Pool, QuantSeq FWD FFPE, Luthor HD and Luthor HD-Pool, are specified to enable correct sample demultiplexing and should not be extended. Longer read lengths for Read 1 for SR or PE formats may be possible depending on the sequencing kit used, and can be implemented at user’s discretion.

⁴ Use Standard Polony Density mode when sequencing new library types for the first time. After loading is optimized, or sufficient run quality and yield is established, High Density (HD) mode can be used to further increase the run output. Effective use of HD Mode has been reported by Lexogen users.

Unless otherwise specified use of the PMG shift is not required. We recommend using default PMG settings (cycles 1-4), as Lexogen libraries retain sufficient nucleotide diversity at the start of read 1. If you wish to optimize, or have optimized PMG shifts for any of your runs, please get in touch with us at support@lexogen.com.

⁵ When sequencing only QuantSeq REV libraries in a whole sequencing run you must use the CSP🔴 provided in the QuantSeq REV V2 kit as a custom sequencing primer for Read 1. IMPORTANT! Use the Replacement Custom Primer workflow from Element Biosciences. This workflow requires an additional reagent from Element Biosciences, either: Custom Primer Set Read 1, Cloudbreak Freestyle (820-00038), or Custom Primer Set Cloudbreak Freestyle (820-00025). The CSP🔴 should not be used, only if QuantSeq REV libraries are mixed with other Lexogen library types at <=25% of the total lanemix. If required, additional aliquots of CSP🔴 can be requested, please contact support@lexogen.com. See also What is the CSP and how do I use it? for further information.

⁶ Relative loading concentration for QuantSeq REV libraries included at <=25% of lanemixes, comprising Lexogen libraries of similar average sizes. NOTE: Sequencing mixed library types in the same lane/run is inherently challenging, requires careful optimization for specific lanemix conditions, and therefore is generally not recommended. This advice is provided for QuantSeq REV as a guideline only. Customers choosing to run mixed lanemixes do so at their own risk. For further information see: Can I pool Lexogen libraries with other library types in the same lanemix for sequencing?.

⁷ Recommended if the aligned insert lengths are <100 bp (i.e. for miRNA, piRNA, tRNA), corresponding to full-length average library sizes (insert + adapters) of <250 bp. For further information on short insert recipe use cases and recommendations see this article from Element Biosciences.