Mate Pair and Paired-End Sequencing – Illumina

Hi folks,

 

 

Today I will post my recurring question about the differences on Mate Pair and Paired-End Sequencing technologies used in Next-Gen sequences.

 

Unfortunately because of technology limitations you can not read long reads, just it their ends (from 35 to 100 bp depending of the chemical and sequencer), to workaround this serious limitation, it was designed this two methodologies.

 

First of all my sources was the own Illumina Website, here is the link for Mate Pair and the link for the Paired-End Seq.  As appear in the website and many articles, these technologies are very useful when you deal with De Novo Sequencing (Assembly a entire Genome) or repetitive parts of genome, why once you have a well aligned sequence and you know the distance between the two sequences you can use the first one as a anchor to determine the second sequence position.  I think the easiest one, is the Paired-End so I will start with it.

I recommend looking at this video for those who know nothing about Illumina technology.

 

Paired-End

 

Paired-End sequecing

It is a modification of the shotgun sequencing(where your sequences have no pairs)

Once you have the DNA fragmented in 200-500 bp, you add adapter in both ends of the sequence of interest (A1 and A2),

In the forth step you generate clusters (spots on flowcell of  same sequences  made by amplification).

Finally after the cluster generation you go to sequencing step (fifth and sixth steps) where using modified dNTPs and primers for know sequences (SP1 and SP2) you read the reads by light signals.

Because you know in the preparation you made sequences of know distance you can/must input this information in your aligner or assembler (depend on your application), because its a very helpful information that will make these softwares to make less mistakes.

Remember that the orientation of a pair of reads (R1/R2) must appear in the aligner output like

(→←) respectively.

 

 

 

 

 

 

 

 

Mate Pair

Mate Pair sequecing

In the mate pair the sequence fragmentation is made in bigger fragments (2-5 kb).

A addition of a Biotin in each 5′ ends is done (step 3).

The sequence with correct addition of Biotin will circularize and after a wash, the sequencing with non-circularized fragment will be thrown away (step 4)

In step 5 and 6, the circularized fragments will be cutted with the biotin in the middle and size-selected (400-600 bp).

And than the sequencing is done normally: adapter with primer sequence addition (step 7), the fragments will be spoted and clutered (step 8), and sequencing (step 9 and 10).

Because you know in the preparation you made sequences of know distance you can/must input this information in your aligner or assembler (depend on your application), because its a very helpful information that will make these softwares to make less mistakes.

Remember that the orientation of a pair of reads (R1/R2) must appear in the aligner output like

() respectively.

 

 

 

 

 

 

 

 

 

 

 

 

Thats it folks, I hope you enjoyed.

 

Bye

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