L******e
发帖数: 679 | 1 What is the difference between 'mate-paired' and 'paired-end' sequencing.
I am totally confused when read papers. Are they the same meaning but
different words?
Thanks. | W*******a
发帖数: 1769 | 2 You need to understand how Illumina seq works. Two ends of the
library molecule anneal to two oligos on the slide, forming
a bridge, and a PCR is performed in situ to amplify this molecule
into a cluster. The efficiency and consistence of cluster forming
is limited by the size of the insert, you can't have very long
product (>1kb)
classical pair-ends are generated directly from short inserts
usually less than 1kb, so no special library preparation method is
required.
For mate-paired, the two ends are usually far apart, 2-5kb, you
can't amplify such long sequences on Illumina chip directly. To
sequences these long inserts, you need to convert them into
short mate-paired libraries basically by removing the sequences in
between. Now the two far apart ends are joined and can be sequenced
by standard Illumina or SOLID chemistry
sequencing.
【在 L******e 的大作中提到】
: What is the difference between 'mate-paired' and 'paired-end' sequencing.
: I am totally confused when read papers. Are they the same meaning but
: different words?
: Thanks.
| L******e
发帖数: 679 | 3 I am going to use SOLiD system.
There is two libraries i am going to prepare:
1), Regular fragmentation, 150 bp / fragment, then link to adaptors.
2), Mate-paired: link two ends of a fragment (1-2 kb) together. (i had
thought that is why also called 'paired-end') .
According to your description, pair-ends is the regular fragments, but why
was call 'pair-ends', is that because it was sequenced from two ends?
Thanks | W*******a
发帖数: 1769 | 4 yes, because when Illumina first developed the seq platform, the
default is to sequence from just one end of the molecule with one
primer. They soon came up with the pair end module, allowing two
seq rxns to be performed sequentially and get sequences from both ends.
PE is backward compatible with single end seq, so nowadays most kits use
PE primers, and you have the option to sequence just one end of it.
I think mate-paired gets its name because you 'mated' the two
ends together, so in the end product they are next to each instead.
Pair-end, from what I understand, means the two reads come from the
ends of the same molecules, so they are 'pairs'. It is just a trivial
terminology thing.
had
but why
【在 L******e 的大作中提到】
: I am going to use SOLiD system.
: There is two libraries i am going to prepare:
: 1), Regular fragmentation, 150 bp / fragment, then link to adaptors.
: 2), Mate-paired: link two ends of a fragment (1-2 kb) together. (i had
: thought that is why also called 'paired-end') .
: According to your description, pair-ends is the regular fragments, but why
: was call 'pair-ends', is that because it was sequenced from two ends?
: Thanks
| m***T
发帖数: 11058 | 5 基本的理解是正确的。
1)pair-end(PE)用的是regular frag lib，但是两头读（分别称为F3和F5），但读的还
是同一片段；
2)mate-pair(MP）则是因为在lib construction过程中用了circulization而且有
internal adaptor(IA)，所以是两个片段(分别称为F3和R3)中间被IA分隔开，实际上读
取的是两个片段。
画个简单的示意图：
PE: ACGTTTCGA........GTACCTA
F3-> <-F5
MP: ACGTTTCGA........GTACCTA-Internal adapter-ATCGGCTA.......
F3-> R3->
why
【在 L******e 的大作中提到】
: I am going to use SOLiD system.
: There is two libraries i am going to prepare:
: 1), Regular fragmentation, 150 bp / fragment, then link to adaptors.
: 2), Mate-paired: link two ends of a fragment (1-2 kb) together. (i had
: thought that is why also called 'paired-end') .
: According to your description, pair-ends is the regular fragments, but why
: was call 'pair-ends', is that because it was sequenced from two ends?
: Thanks
| L******e
发帖数: 679 | 6 Many thanks to you two . It is quite clear for me now. | e*******e
发帖数: 1837 | 7 很清楚.这个应该马一个了吧.
【在 m***T 的大作中提到】
: 基本的理解是正确的。
: 1)pair-end(PE)用的是regular frag lib，但是两头读（分别称为F3和F5），但读的还
: 是同一片段；
: 2)mate-pair(MP）则是因为在lib construction过程中用了circulization而且有
: internal adaptor(IA)，所以是两个片段(分别称为F3和R3)中间被IA分隔开，实际上读
: 取的是两个片段。
: 画个简单的示意图：
: PE: ACGTTTCGA........GTACCTA
: F3-> <-F5
: MP: ACGTTTCGA........GTACCTA-Internal adapter-ATCGGCTA.......
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