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A comparison of pooled and barcoded approaches
for DNA sequencing of large-insert clones
from metagenomic libraries
Kathy N. Lam
Supervised by
Trevor C. Charles
Function-based meta-genomics
to generate metagenomic libraries...
Clone metagenomic DNA...
to screen based on function.
Function-based screens = lots of clones!
lactose utilization*
xylose utilization
cellobiose utilization*
antibiotic resistance
bacterial conjugation
Goal: obtain DNA sequence of large-insert cosmid clones
Ba
rcod
ed
seq
uenci
ng P
oole
d se
quencing
Sequence 92 cosmid clones, both ways.
Compare pooled sequencing results to barcoded results,
using barcoded results as a reference.
Analyze 74 clones:
1. Coverage
2. Identity
3. Cost
Pooled vs. barcoded: coverage
WITH KATJA ENGEL AND GREG VEY
Pooled sequencing works! ... but here, with incomplete coverage.
Accounting for unretrieved sequences from the pool
Pooled sequencing worked better than we thought! But we need more depth to close the gaps.
WITH GREY VEY AND MICHAEL HALL
Pooled vs. barcoded: identity
Pooled sequencing generates accurate sequence data; no problems with chimeras.
WITH KATJA ENGEL AND GREG VEY
Pooled Contig
Barcoded Contig
Pooled vs. barcoded: cost
Pooled sequencing requires optimization (sequencing depth), but is more cost-effective.
Summary
Compared barcoded sequencing to pooled sequencing
Pooled sequencing results in accurate sequence data
Function-based screening of metagenomic libraries yields many
positive clones to be sequenced
Pooled sequencing coverage would likely match that of barcoded with
increased sequencing depth
Acknowledgements
Supervisory Committee
Trevor Charles
Josh Neufeld
David Rose
Gabriel Moreno-Hagelsieb
Charles Lab
Everyone, esp.
Katja Engel
Greg Vey
Jiujun Cheng
Tanya Romantsov
Cveta Manassieva
Neufeld Lab Michael Hall