Mate-Pair library consists of approximately 150-300 bp fragments. They are composed of 2 DNA segments originally located 2-5 kb apart in the genome of interest. With a mate-pair library it is therefore possible to span gaps or repeats of up to 2-5 kb.
For library preparation genomic fragments are sheared and size selected for approx. 3.5 kbp fragments. The fragments are end labelled with biotin and circularised. The circularised molecules are sheared once more and the library is enriched for biotin labelled fragments. Sequencing these biotinylated fragments generates the desired 'outward-facing' paired reads, meaning they align to a reference sequence outward-facing from each other. These outward-facing reads align to the reference sequence with a gap size of 2-5 kbp.
The mate pair library has several restrictions, limiting the usefulness of the library for high quality scaffolding of contigs:
1) Inward facing reads: Unbiotinylated fragments that have not been successfully washed away will cause undesired inward facing reads (see Illumina’s mate pair sample preparation guide). These inward-facing reads are like shotgun paired end reads as they align to the reference with a smaller gap size of around 200-300 bp.
2) Genomic shearing of the circularized biotinylated molecules is a mechanically process and will produce fragments having the biotin label in the middle of the fragment, but also on all other positions of the fragment. When sequencing fragments having the biotin label (and thus the crossover) in the middle of the fragment the desired outward-facing reads will be generated. However, when sequencing fragments having the biotin and the crossover at the outer 100 bps of the fragment, one of the resulting reads will be a hybrid read. The hybrid read will contain sequence from one AND the other end of the original 3.5 kbp fragment and there is no information about the location of the changeover. These read pairs are worthless for the assembly and can even lead to misassemblies.
For high quality high-throughput scaffolding by Illumina sequencing Eurofins Genomics offers an alternative proprietary library, the so called long jumping distance libraries (LJD's) with jumping distances of 3 kbp, 8 kbp, 20 kbp or 40 kbp. Read more