2013 UW Grad Conference

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.

A function-based screen: lactose utilization

Function-based screens = lots of clones!

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.

Pooled vs. barcoded: coverage

WITH GREY VEY AND MICHAEL HALL

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

Thank you.