Genomic Sequencing
Very Nice Tutorial by Arthur Gruber
NCBI Genomic Sequence Assembly and Annotation Process
•Large region purified and broken into 100 kb or slightly
larger random fragments •Fragments
cloned into vectors: eg. yeast artificial chromosomes (YACs) or bacterial
artificial chromosomes (BACs)
•library of clones screened for overlapping contigs
•Once map obtained, unique overlapping clones chosen for sequencing
•Clones too large for direct sequencing
•Clone
is broken down into subclones, with some level of redundancy (typically
4x – 10x
coverage)
•Subclones (500 bases) are sequenced
•subclones assembled based on overlapping sequences (and mapping information)
Shotgun sequencing
•Map data is ignored
•Both ends of DNA fragments of short (2kb), medium (10 kb) and long (100
kb) fragments sequenced
•End sequences used as anchors
•Genome randomly broken up into small (500 base) pieces which are then
sequenced
DNA Sequence assembly
Try to assemble the four fragments below:
cacaaaatataagaaggaaaagtgctgatt
tgctgattttcctacctttgtgtttcatga
cttaaaaccaatgtctcataaatcatgaaa
gttttaagcaaaactgagttagagcaactc
cacaaaatataagaaggaaaagtgctgatt
tgctgattttcctacctttgtgtttcatga gttttaagcaaaactgagttagagcaactc
TTTCATGATTTATGAGACATTGGTTTTAAG
reverse complement ^
cttaaaaccaatgtctcataaatcatgaaa
Required overlaps for contig assembly usually at least 20 bp up to 100 bp
Genome assembly Programs
•PHRAP - most commonly used
•Arachne
•CAP3
•GigAssembler
•Phusion