Question about Blocker in Paired-seq

[minglu-xie]

Hello Dr. Chen,
Thank you for sharing detailed information about single-cell sequencing technologies! I’ve been studying the paired-seq method and have a question regarding blocker design.
In the Paired-seq protocol, why are linker-specific blockers essential? Could you clarify their role in preventing unwanted ligation events (e.g., linker self-ligation or dimerization), and how their absence might compromise library integrity? My understanding is that sticky-end ligation is inherently specific, so I’m unclear why blockers are critical here.

Thank you for your time and help.

Best,
Minglu

[dbrg77]

Hi Minglu,

Thank you for your questions.

I don't really know the exact reasons about this design, but it seems all split-pool ligation based methods (e.g. SPLiT-seq, Paired-seq, SHARE-seq) have this step to block the linker during ligation.

My own understanding is that the purpose is not really to prevent self-ligation or dimer etc. Instead, it is mainly used to reduce cross contamination among different wells. Since the ligation reaction is never 100% complete, there will always be some free floating linkers and barcodes. If the linker is not blocked, when pooling different wells together, the linker may bring barcodes from unintended wells.

I will think about this as well and draw some graphs to help visualise this problem when I have time later this month.

Regards,
Xi

[minglu-xie]

Thank you so much, Xi, for the detailed explanation — that makes a lot of sense, especially the point about minimizing cross-contamination between wells due to incomplete ligation.

I had a follow-up question: whether the blocker could potentially outcompete the unligated, annealed linker-barcode complex during the pooling and blocking steps?

Looking forward to your visualizations whenever you have time — that would definitely help clarify things further!

Best,
Minglu