BioMicroCenter:Element Sequencing

Element Sequencing

The Center currently hosts an AVITI24 platform from Element Biosciences. The AVITI24 platform supports both sequencing and cytoprofiling. For most sequencing needs, the Center will use Element's Cloudbreak chemistry kits. Cloudbreak is a sequencing-by-binding method that uses the concept of avidity to produces high quality sequencing reads. Cloudbreak circularizes linear libraries, and then performs rolling circle amplification to generate thousands of copies of the original template on a single, linear strand. The strand is then packaged up into what is known as a polony. These polonies are washed with a complex of primers, polymerase, and dye-labelled avidites. Avidites are multivalent-nucleotide-ligands on dye labeled cores capable of binding to multiple sites to form a polymerase–polymer–nucleotide complex within a polony. After imaging, the avidites are washed away and the growing read strand incorporates a single nucleotide that is reversibly blocked on the 3' end. The polonies are again washed with avidites, repeating the process over many cycles. The AVITI24 can run 2 flow cells per run, each hosting 2 independently-addressable lanes. Note, however, that the reagents that come with each kit address the entire flow cell. In other words, while the flow cells may have 2 different libraries loaded, one in each lane, both lanes will be washed with the same set of primers. Element currently offers kits with 150 cycles and 300 cycles, both supporting 2 lanes, as well as a 600 cycle kit that supports a single lane. With Cloudbreak's update to "Freestyle", the AVITI24 is capable of sequencing both Illumina-ready and/or Element ready libraries directly on the flow cell. High-output kits generally produce 300 to 800 million high quality read. Index hopping rates are virtually nil, limiting the need for UDI's. The AVITI24 can also perform single cell spatial imaging of RNA, proteins, phospho-proteins, and cell morphology using Teton chemistry for cytoprofiling.

Workflow

Library prep options for DNA or RNA are available for Element sequencing. Once Element-ready or Illumina-ready libraries have been submitted, we will verify fragment size via AATI's Fragment Analyzer or Femto Pulse, and quantify the final loading pool via qPCR and Qubit. We can only guarantee minimum reads per lane if: a)the BMC has performed quality control, b)the samples have high diversity, especially within the first 5 bases of read 1, and c)submitted libraries are at least 2 nM.

Requirements/Things to Consider

1. Polony-mapping-generation (PMG), is done on the first 5 reads of read one. You will want to have AT LEAST these 5 positions with high diversity (roughly equal A,G,C, and T's across the flow cell per read). However, the AVITI24 does give us the option to PMG off index 1 (sp27), with a suggested multiplex of 64 or more. PMG can also be done upstream of the start of read 1, up to a total of 20 bases. However, these bases will be read as dark. You will not received data on these bases, and you will lose those cycles. If these options can not be applied, then we will need to increase the percent phiX control spiked into the final loading sample by as much as 30% to increase the read 1 diversity.
2. Illumina-ready-linear libraries (have p5/p7 flow cell binding sites) are preferred as these tend sequence much more efficiently on the Freestyle flow cell, and can be applied to other platforms should the need arise.
3. Element-ready-linear libraries (sp5/sp27 flow cell binding sites) must have a 5'-phosphate on the sp5-end sequence in order to circularize on the AVITI. If you choose to use Element's flow cell binding sites, sp5 and sp27, it's suggested that library prep primers are HPLC cleaned and used within a month or so as any truncated primers, as part of the primer creation process or due to degradation over time, are unlikely to circularize leading to a decrease in read count.
4. Library preparations involving biotin-modifications, poly-A tailing, and/or bead-based normalization will not sequence well, if at all.
5. Read direction will be just like MiSeq. The index reads are sequenced first, starting with the sp27 index (which will be read off the sequencing primer site), followed by the SP5 index (which will be read off the sp5 flow cell binding sequence). The read sequences are then read starting with read 1 (using the Read 1 primer sequence), followed by the amplification of the of the reverse strand. Read 2 is then read using the reverse complement of the Read 2 primer sequence.

   Data Handling

   Custom Sequencing

   Service Element Sequencing SUBMISSION Illumina OR Element libraries MIN VOLUME 15 uL MIN CONCENTRATION 2 nM INCLUDED SERVICES Quality Control:Fragment Analyzer and qPCR Element Sequencing Demultiplexing ADDITIONAL SERVICES Quality Control Element library preperation DATA FORMATS FASTQ (stored 1 year) BCL (stored 30d) QUALITY CONTROL FASTQC Basic run metrics (alignment rate, complexity) Basic RNAseq metrics (where applicable) Basic paired end metrics (where applicable) Contamination checks PRICING LINK SUBMISSION MIT - ilabs The AVITI24 Platform AVITI NOTES SEQUENCER READS/FLOWCELL Low number is minimum per lane for standard Illumina libraries. 300M HQ read pairs PF/flowcell RUN TIME 75 PE 24 hours 150 PE 38 hours 300 PE 60 hours KITS AVAILABLE 2x75 Cloudbreak High 2x150 Cloudbreak High 2x300 Cloudbreak High - Single end supported - each kit comes with 34 additional cycles KNOWN APPLICATIONS Whole Genome Sequencing RNA Sequencing 16S Single-cell Target Sequencing and Enrichment Metagenomics KEY NOTES 4-color chemistry Patterned flow cell 2-lanes per flow cell Q40+ > 80% Low-binding surface chemistry Adept Workflow for adaptation of Illumina libraries THANKS TO Element Biosciences