1. 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
6. 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
7. Barcodedsequencing
Pooledsequencing
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
8. Pooled vs. barcoded: coverage
WITH KATJA ENGEL AND GREG VEY
Pooled sequencing works! ... but here, with incomplete coverage.
10. 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
11. Pooled vs. barcoded: identity
Pooled sequencing generates accurate sequence data; no problems with chimeras.
WITH KATJA ENGEL AND GREG VEY
Pooled Contig
Barcoded Contig
12. Pooled vs. barcoded: cost
Pooled sequencing requires optimization (sequencing depth), but is more cost-effective.
13. 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
