There are a number of factors that need to be taken into consideration when constructing libraries. If you are preparing to send a company your premade libraries, this article will provide some tips and tricks for this preparation so that you get the best results from your sequencing. This article will also discuss common issues that can arise during premade library sequencing and how to avoid or rectify them.
Library preparation consists of 4 steps:
- Fragmentation: samples must be fragmented into uniform-sized pieces within a range suitable for the chosen sequencing platform.
- End repair & A-tailing: an adenine base is added to either end of the newly generated fragments. This overhang facilitates the addition of adapters with a thymine overhang.
- Addition of adapters: a ligase enzyme covalently binds adapters to the fragments, producing a full library compatible with binding to the flow cell of the sequencing instrument.
- PCR amplification: if it's low-input samples, PCR amplification will be required.
Fragmentation
There are two important factors to consider with the fragmentation process – the method used and size selection. Most sequencing instruments work best when supplied with fragments of a similar size. Three methods exist for fragmentation – physical, chemical, and enzymatic.
• Physical: considered a "clean" method as it doesn't introduce any additional chemicals or enzymes to the system. This is the primary method used by service providers as it requires specialised equipment, so is not readily accessible to individuals preparing their own libraries.
• Chemical: mostly used for preparing RNA libraries; however, it can introduce cations to the system which may interfere with the downstream sequencing process.
• Enzymatic: most commonly used for in-house library preparation as it only requires standard lab equipment and is highly scalable. It requires a low input for PCR free library generation; however, it can cause fragmentation bias and the ratio of enzyme to substrate (nucleic acid) needs to be controlled for good results.
Size selection is important for eliminating sub-optimal fragments from the library before introducing them to the sequencing platform. It ensures the fragments are within the optimal range for the particular instrument in use.
This step also minimises wastage of sequencing capacity. Fragments that are too short will result in the formation of primer dimers. If fragments are too long, the machine won' t be able to cover the length of them or produce any useful sequencing data. Two methods exist:
Gel-based: a reliable method but is low throughput and can eliminate only very small or very large fragments.
Magnetic bead-based: the most common method used in commercial kits. This method is cost-effective with higher throughput and higher size separation resolution.
Anatomy of a Library
Careful library preparation is crucial for successful sequencing and data analysis. The purpose of library preparation is to make the DNA fragments that are to be sequenced compatible with binding to the flow cell. After fragmentation, adaptors are added to either end of the generated fragments to allow them to bind to the flow cell and be sequenced.
The adaptors added to the fragments consist of three elements on either end - a P5 or P7 binding region, a read sequencing primer binding site, and an index sequence.
• The P5 and P7 binding regions are complementary to the flow cell oligos and allow for hybridisation and cluster generation.
• The index regions are short sequences of 6-10 base pairs that allow for multiplexing i.e. for sequencing multiple samples in one run, and identifying them afterwards. This example has an index on either end and is therefore a “dual index” library.
• The read 1 primer binding site is where the forward read primer for sequencing binds and the read 2 primer binding site is where the reverse read primer binds. A "single-end" library will contain only the read 1 primer binding site, whereas a "paired-end" library will contain both read 1 and read 2 primer binding sites. This allows for forward and reverse sequencing for higher coverage.
I hope you enjoyed this content, and now feel ready and confident to learn more about the Library Quantification and Quality Control in our next post, stay in tune!
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