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Old 03-08-2010, 05:54 AM   #1
tccbrm
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Smile Question about solexa sequencing

Hi all,

I am a new here, hope everyone can have a nice day!

Here is my question, before sequencing, we have to make some libraries in different insert size. Is that the short insert size (200 ~500 bp) are using single-end sequencing for the alignment of contigs, and the long insert size (over 2kb) are using paie-end sequencing for joining the contigs into scaffolds?

Thanks in advance

Man
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Old 03-08-2010, 11:27 PM   #2
simonandrews
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If you're using the Illumina platform for sequencing then your choice of insert size is limited by the technique used to generate the clusters on the flowcell. Generally your inserts have to be <800bp to produce usable clusters so you don't really have the option to use longer inserts.
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Old 03-08-2010, 11:32 PM   #3
tccbrm
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So, if I want to sequence a species, is it neccessary to senquence the single-end or what I can do is just the pair-end sequencing then align the reads?

Thanks
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Old 03-09-2010, 03:40 AM   #4
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Quote:
Originally Posted by simonandrews View Post
If you're using the Illumina platform for sequencing then your choice of insert size is limited by the technique used to generate the clusters on the flowcell. Generally your inserts have to be <800bp to produce usable clusters so you don't really have the option to use longer inserts.
This is true for the Illumina paired-end protocol, in which you sequence both ends of a contiguous stretch to DNA. But Illumina also has the mate-pair protocol which allows you to get paired reads separated by 2-5kbp.
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Old 03-09-2010, 01:05 PM   #5
drio
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Quote:
Originally Posted by kmcarr View Post
This is true for the Illumina paired-end protocol, in which you sequence both ends of a contiguous stretch to DNA. But Illumina also has the mate-pair protocol which allows you to get paired reads separated by 2-5kbp.
Anyone cares to talk about its experience with that protocol? How stable is it?
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Old 03-09-2010, 01:31 PM   #6
kmcarr
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Quote:
Originally Posted by drio View Post
Quote:
Originally Posted by kmcarr View Post
This is true for the Illumina paired-end protocol, in which you sequence both ends of a contiguous stretch to DNA. But Illumina also has the mate-pair protocol which allows you to get paired reads separated by 2-5kbp.
Anyone cares to talk about its experience with that protocol? How stable is it?
We have done it once and it mostly worked as expected. That is to say the vast majority of reads were properly paired and spaced ~2.8-3.0 kbp apart. However we observed and extreme bias in the nucleotide composition at the start of the reads. I posted about this in another thread (see here) but unfortunately got no hits there. I to am very interested in hearing what other's experiences have been with the mate-pair protocol.
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Old 03-09-2010, 02:32 PM   #7
drio
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Quote:
Originally Posted by kmcarr View Post
We have done it once and it mostly worked as expected. That is to say the vast majority of reads were properly paired and spaced ~2.8-3.0 kbp apart. However we observed and extreme bias in the nucleotide composition at the start of the reads. I posted about this in another thread (see here) but unfortunately got no hits there. I to am very interested in hearing what other's experiences have been with the mate-pair protocol.
I'll get back to you on that.. I have to double check.
Ping me if I don't reply in the next couple of days.
I'll reply to your other post.
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