Forum:shotgun sequencing and clone-based sequencing

Question

[mm521]

For WGS sequencing, the DNA was sheared first and then cloned into a vector, finally assembly contig and PCR gaps.

1. I found clone is used in shotgun method. So cloned based sequencing method is a part of shotgun sequencing or a separate method? Are they supplement each other or independent method?
2. In shotgun sequencing, after cloning, DNA is sequenced and show overlaps .But how exactly the DNA is sequenced within vector?

Thanks

Answer 1

[Devon Ryan]

1. It doesn't have to be, but practically speaking it's a convenient way to do so. One could alternatively use random primers to get the sequencing adapters on the end of the fragment. Then you need to amplify single fragments, for which one could think of a couple ways. Honestly though subcloning is fairly convenient.
2. The cloning vector has primer sites flanking the insert. These are used with normal Sanger sequencing.

[mm521]

1: The sheared smaller segments are from a number of same copies of DNA or one DNA molecule? 2: There is a term called "single stranded region" for assembly step. Does that mean the region with overlap from only one-direction read, or means that this region is not overlapped.

Thanks.

[Devon Ryan]

You perform shotgun sequencing from a large amount of DNA, there are no single copies of anything at the beginning. I have no idea what you're talking about with "single stranded region". I can come up with multiple things that that could refer to.

Answer 2

[h.mon]

My understanding is there is pure shotgun sequencing, where you directly shear into small fragments and sequence those small fragments; and hierarchical shotgun, when you first clone big fragments about which you have mapping information, then shear those clones into small fragments and sequence then, assemble the smaller fragments for each clone separately, finally assemble the clone contigs together.

[mm521]

I have a question for you said hierarchical shotgun. When you make clones, it always related to a vector, insert into a plasmic or something, then when you shear the clones, it will be a mix of your fragments and vector fragments. So how to make the assemble is from the sequence we want?

Thanks

[GenoMax]

Since you know the sequence of the vector you can subtract that out once you create an assembly for the clone in question. You can precisely sequence the ends of the inserts. Original human genome sequence was done by this method (see Figure 2).

10x genomics uses somewhat similar (but modern) technology for sequencing.