iSeq 100 user new here

[100%Wool]

Hi Folks,
iSeq 100 user here, checking in!
May all your runs be bountiful
Wool.

[Zed]

Hi Everyone, We acquired this machine recently. we are resource-limited researchers and want to sequence the CoV-2 genome from a limited number of patients. How many iseq cartridges will be needed per sample, if we use Enrichment Sequencing by Spiked Primer MSSPE method?

[jjimenez]

Hello Everyone,
I know this thread is quite old, but wanted to know how has your experience been so far with the iSeq 100?

We have only performed one run, and although Q30 was quite good for the Read 1 and 2, the indexes were read with lower Q30, even though we had a good balance of bases in each cycle (checked after the run in basespace).

I was wondering if there were other tips to improve Q30 during indexing? It's a shame to lose good reads due to poor indexing

[Ben3]

jjimenez I would imagine that the only way to improve the Q30 for index reads is to further increase the index complexity or add more PhiX. You said you had a good balance but I wondered if there was a way you further increase the diversity of these bases.

[jjimenez]

OK,
So this was not the greatest SeqRun, a lot of short fragments that give rise to the Poly-Gs you see after Cycle 120 (clearly, we have to be more stringent with the size selection). Going back to the Q30 metrics

Read 1: 92.75% >Q30
Read 2: 82.71% >Q30
Read 3: 79.38% >Q30
Read 4: 90.02% >Q30


During the indexing reads, no base goes above 40% representantion. I wouldn't think its a diversity issue? And PhiX wouldn't help, since its not indexed (unless we get the indexed one from other sources, but I dont think we'll do that)

The run was a bit underloaded though (82% ocupation), I wonder if that has any effect.

[Ben3]

jjimenez you're right, for some reason, I kept thinking PhiX adds to the index diversity but it doesn't. Although having a slightly underloaded run shouldn't cause this issue either. I haven't run anything on the iSeq yet, but whenever I had this problem on the NextSeq, I would just spike in other libraries for additional diversity. I know that doesn't answer your question but it seemed to help.