Selecting indexes that give a good discernible signal on the Illumina platforms can very much help increase the reads successfully assigned to each sample in a pool. There may also be issues that pools that demultiplexed well on the older instruments (NovaSeq 6000) may not be as good on the newer sequencers (NovaSeq X, NextSeq 2000) due to how these machines work.
There are full guides to indexing choices on the Illumina web site.
We recommend you read these guides to understand the issues involved. Illumina also provide a document "Index Adapters Pooling Guide"that gives a fuller read of the subject.
We have developed a tool to help visualise how your chosen barcodes will perform before you commit to assigning the labels to your actual samples at the bench. This tool is part of the submission portal.
The purpose of this tool is to allow you to see if your choice of barcodes will perform satisfactorily on the Illumina instruments. It will help visualise the amount of signal you would expect if all barcodes are present equally, and crucially if there are too many cycles where the signal may be insufficient or otherwise go against Illumina's guidance. It will display any potential issue that is found for all the cycles.
You can copy and paste your sequences into the text box on the page to generate the plot, or you can prepare a file with the sequences and upload that when you are ready. The tool accepts both raw sequences and/or the barcode labels present in our service. You can upload a one or two column CSV or Excel .xlsx file with the sequences, or the tool will also accept our SLX submission form for checking.
Uploading the submission form to this tool does not make a submission.
You need to submit sequences of the same length: it will not be able to analyse a mix of shorter and longer indexes.
This section is relevant for the single index, four barcodes per sample 10x systems, namely SIGA and SINA.
If you submit a file with SIGA or SINA index labels, or a CSV with four columns, the tool will automatically convert these into single indexes with four times the number of barcodes. This is because these kits apply four single indexes to every sample, so each one of them adds to the combination of the single index read.
For example, if you submit a file containing:
SINAA1 SINAA2 ACGTACGT,CGTACGTA,GTACGTAC,TACGTACG
the index combination is:
AAACGGCG CCTACCAT GGCGTTTC TTGTAAGA AGCCCTTT CAAGTCCA GTGAGAAG TCTTAGGC ACGTACGT CGTACGTA GTACGTAC TACGTACG
These are the four sequences of the first label plus the four sequences of the second label and the four sequences provided just as sequences. You will see in the text area on the page this has happened as well as a plot with eight i7 sequences.