US 5,670,321 AGranted

Efficient method to conduct large-scale genome sequencing

AssigneeMercator Genetics, Inc.

InventorsBruce E. Kimmel, Michael Ellis, David Ruddy

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Abstract

An efficient method for sequencing large fragments of DNA is described. A subclone path through the fragment is first identified; the collection of subclones that define this path is then sequenced using transposon-mediated direct sequencing techniques to an extent sufficient to provide the complete sequence of the fragment.

Claims4

Dependent claims

A method to determine the nucleotide sequence of a fragment of DNA, said fragment having a length of more than about 30 kb, which method comprises:

(a) providing said fragment in a first host cloning vector;
(b) shearing a composition comprising said vector containing the inserted fragment under conditions to obtain subfragments of approximately 2-5 kb;
(c) inserting each of said subfragments into a second host cloning vector to obtain a first library of subclones;
(d) recovering sufficient subclones from said first library of subclones to provide 1.times. coverage of the fragment when about 400 bases at one end of each of said subfragments is sequenced and about 7-8.times. coverage of the fragment when the complete nucleotide sequence of each of the subfragments are determined;
(e) sequencing about 400 bases at a first end of each of said subfragments in the recovered subclones to obtain first end sequence information;
(f) placing the first end sequence information into a searchable database;
(g) searching said database to identify subclones containing subfragments that include nucleotide sequences that match vector sequences present in the first host cloning vector;
(h) recovering the identified subclones;
(i) sequencing about 400 based at a second end of the subfragment of each of the identified subclones, said second end opposite from the first end sequenced in step (e), to obtain second end sequence;
(j) searching the database to identify subclones containing subfragments that include nucleotide sequences that match said second end sequence;
(k) recovering the identified subclones;
(l) sequentially repeating steps (i)-(k) until a collection of subclones has been identified that represents the complete fragment;
and
(m) obtaining a sufficient portion of the complete nucleotide sequence of the subfragment contained in each of the subclones from the collection, using transposon-mediated sequencing, to obtain the complete sequence of the fragment.

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Dependencies are inferred from the claim text.

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Abstract

A method to determine the nucleotide sequence of a fragment of DNA, said fragment having a length of more than about 30 kb, which method comprises:

(a) providing said fragment in a first host cloning vector;
(b) shearing a composition comprising said vector containing the inserted fragment under conditions to obtain subfragments of approximately 2-5 kb;
(c) inserting each of said subfragments into a second host cloning vector to obtain a first library of subclones;
(d) recovering sufficient subclones from said first library of subclones to provide 1.times. coverage of the fragment when about 400 bases at one end of each of said subfragments is sequenced and about 7-8.times. coverage of the fragment when the complete nucleotide sequence of each of the subfragments are determined;
(e) sequencing about 400 bases at a first end of each of said subfragments in the recovered subclones to obtain first end sequence information;
(f) placing the first end sequence information into a searchable database;
(g) searching said database to identify subclones containing subfragments that include nucleotide sequences that match vector sequences present in the first host cloning vector;
(h) recovering the identified subclones;
(i) sequencing about 400 based at a second end of the subfragment of each of the identified subclones, said second end opposite from the first end sequenced in step (e), to obtain second end sequence;
(j) searching the database to identify subclones containing subfragments that include nucleotide sequences that match said second end sequence;
(k) recovering the identified subclones;
(l) sequentially repeating steps (i)-(k) until a collection of subclones has been identified that represents the complete fragment;
and
(m) obtaining a sufficient portion of the complete nucleotide sequence of the subfragment contained in each of the subclones from the collection, using transposon-mediated sequencing, to obtain the complete sequence of the fragment.

11 more elements