Submission Form Guidelines for NovaSeq, MiSeq and NextSeq

1. Fill out the SLX identifier which is printed on the tube containing your library (SLX-xxxxx). SLX tubes can be obtained either from CRUK reception or via UMS for University users.
2. Enter the number of samples in the pool to be sequenced. If this is a pool without multiplexing, or a pool with in-line barcodes or custom indexing, set the pool size to 1.
3. Choose a sequencing workflow:
   • NovaSeqX : use this for direct NovaSeq sequencing requests.
   • NovaSeq X with MiSeq Nano QC: use this for NovaSeq sequencing requests which include a MiSeq Nano validation run of your library pool first; this provides an opportunity to adjust your pool if required before committing to the NovaSeq flowcell. This is highly recommended if you want to run multiple lanes of sequencing.
   • MiSeq: use this for all standard MiSeq v3 sequencing requests.
   • MiSeq Express: use this for MiSeq v3 sequencing if you want us to use your quantified concentration. We ask for samples to be submitted at 4nM and will not quantify your library ourselves.
   • MiSeq Nano: Use for MiSeq Nano requests.
   • MiSeq Nano Express: as for MiSeq Express but using a MiSeq Nano kit.
   • NextSeq 2000: use this for all standard NextSeq 2000 sequencing requests.
4. Select sequencing type from the drop-down menu: “Single Read” or “Paired End”.
5. Select read length from the drop-down menu or enter it manually if it is asymmetrical (e.g. 92+26).
6. Enter the number of lanes required. See table 2 below for expected yields for each flowcell type.
7. For NovaSeq and NextSeq 2000 runs, select the flowcell type.
   The NextSeq 2000 flowcells are the “P” ones (P1, P2, P3); The NovaSeq X flowcells are the “B” ones (1.5B, 10B, 25B).
   Leave this field empty for other sequencers.
8. Select library type from the drop-down menu.
   Important: if submitting for the library types “Amplicon low diversity”, “Amplicon high diversity” or “Bisulphite” please read table 3 below regarding PhiX spike-in percentage.
9. If you are submitting for unspecified indexing where you will demultiplex yourself, you must specify the index lengths required for each index. _We cannot accept your library unless we have this information_.
10. Enter the average library length including insert and adapters (see point 4 of further information).
11. Select required option for custom primers. See the custom primers guide for more information.
12. Select sample type, sample source and species from the drop down menus.
13. Complete billing information. This is critical for correct invoicing: please follow the popup instructions in the field. Only enter a PO number if applicable to your department or organisation. Leave PO number blank if it does not apply! Resist the temptation to put “N/A” or “None”: it will complicate your bills.
14. Enter any submission comments you think may be important to your submission (e.g., PhiX % you require spiked into the lane if it is non-standard).

1. Complete a separate row with a sample name and index name for each sample within the pool. For example, a pool of six libraries should use six rows.
   • Sample Name can be anything you choose to represent that sample.
   • Index must be the corresponding Index Name (e.g. i701_i501) rather than the index sequence. Some index types are grouped (for instance all Nextera kits as they all use the same original set of Illumina Nextera index sequence), so as long as the sequence matches your index sequence you are able to submit using that index name.
   • Species is only needed if you have a mix of species in the pool. Use this field to assign a species for any sample whose species differs from that set in cell C25 (the species common across the pool).
2. For a pool with no indexing, inline barcodes or unspecified indexing, complete a single row for the whole pool; i.e. a pool of six libraries is still only one row in the sample information table. The index should read “None” for no index, “INLINE” for inline barcodes and “Unspecified Index” for custom indexing. As noted in point 2, the number of samples in pool should be set to 1.

A full list of the supported indexes can be found on the index information page. If your index type is not currently supported, email the genomics helpdesk to enquire if the new indexes could be added. We cannot add every available index, but we will add to our list if there is a significant requirement for new indexing kits or options.

1. Run your completed submission form through this submission application. Any errors will be reported back to you for correction. If there are no errors, you will be asked to confirm your submission to the Genomics' LIMS system.
2. Once your submission is complete, bring your SLX tube(s) to Genomics lab 026 or the Cambridge Institute reception for our external collaborators. Reception can accept tubes between 09:30-16:30 Monday to Friday.

1. To request resequencing of a previously sequenced library, please contact the helpdesk with your request. Please also copy in your approver in your email if applicable.
2. Average Library Length is used when quantifying your library by qPCR and the accuracy of this field directly affects your sequencing yield. It must be the total length of your library including Illumina adapters. For example, 180 bp insert + Illumina adapters (~120 bp) = 300 bp total length.
   We recommend you measure this for each pool you submit using Agilent's TapeStation or Bioanalyzer (using the region table feature).
3. We strongly recommend that you do not mix barcoding kits (index types) in the same pool. We cannot demultiplex such pools and there is a risk of using barcodes too similar to each other for demultiplexing to separate.
4. If you must submit a pool with two or more different barcoding kits used in it, please submit with Index Type set to “Unspecified (Other)”. The pool can be sequenced to your requirements but it is wholly your responsibility to demultiplex the FASTQ files you will receive. The sequencing service does not support demultiplexing such pools.
5. If you submit a sample for multiple lanes of NovaSeq X, we strongly suggest running a MiSeq i100 to help us optimise loading on the instrument. Failing that, we can't guaranty optimal results.

• MiSeq: 15µl of your pooled library at 10-20nM.
• NextSeq: 15µl of your pooled library at 10-20nM.
• MiSeq Express: 15µl at 4nM precisely.
• NovaSeq submissions dependent upon flowcell type:

Table 1 - NovaSeq X Plus submission requirements (library volume)

Please do not submit molarities above 50nM.

Table 3 - Reads per lane

Illumina specification and performance typically seen in the genomics core.

Table 4 - PhiX % spike-in for certain library types

* Optimisation may be required.