454 sequencing put to the test using the complex genome of barley

BMC Genomics

Table 1 Comparison of ABI-Sanger and 454 sequencing procedures

	ABI-Sanger	454	time required^a
Isolation of BAC DNA	x	x	1 day
Mechanical shearing	x	x	2 h
Cloning	x		4 h
Clone picking	x		2 h
Plasmid DNA extraction	x		20 h
Reactions on thermocycler	x		36 h
Clean up reaction products	x		2 h
ABI 3730xl sequencer run	x		24 h
454 sequencing library		x	4 h^b
Amplification in PCR microreactors		x	6 h^b
GS 20 sequencing run		x	4 h^b,c
Assembly of raw sequences	x	x	days to weeks

^aThe procedures and estimated time requirements describe the process for sequencing a BAC clone with a size of 100 kb. For ABI-Sanger, numbers are calculated to reach an approximately 10-fold coverage.
^baccording to [5]
^cOne 454 GS20 run produces ~20 Mb, approximately 10 times more than required for a sufficient coverage (20 ×) of a 100 kb BAC clone.

ISSN: 1471-2164