Question: PacBio multiplex design
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gravatar for mcombs333
4 days ago by
mcombs3330
mcombs3330 wrote:

Hello Biostars!

I'm planning to use PacBio's Single Molecule, Real-Time (SMRT) method to sequence two genes, each 1-2kb and looking for insight about how to plan out my project.

1) Is there a trade off in the length of target sequence and the number of samples I can fit in a run? For example can you fit twice as many samples if the amplicon is half as big, or some other general rule of thumb?

2) For multiplex sequencing on PacBio's SMRT platform, what is the trade off between the number of barcodes I use and the overall sequencing depth/accuracy? Does anyone have experience deciding how many samples to fit into a run to balance cost with data quality?

I know it may seem silly to use SMRT (which can do 50kb at high accuracy) for sequencing 1.5kb fragments, but it seems better than doing a tiled PCR design to capture the same locus with two short reads (300x300 paired end sequencing) on Illumina. I'm open to other ideas about how to best get high accuracy sequences for 1-2kb amplicons.

Thanks

sequencing pacbio • 41 views
ADD COMMENTlink written 4 days ago by mcombs3330
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