Eisen Lab Blog

Now that we have chosen our candidates we are in the process of preparing libraries for sequencing. I’ve learned a lot about this process in the past few weeks so I thought I’d share some of what I’ve learned.

First, what is a “genomic library” anyway?

“Genomic library” is the term used to describe the prepared genomic DNA that is sent to the Illumina sequencer for sequencing. Library preparation is a critical step because the quality of a library preparation often determines the quality of the sequencing and the ease of assembly.[i]

How does one prepare a genomic library?

Although there are many different methods to choose from in library preparation all methods have the same basic two goals.

1.  To cut the DNA into small pieces. The size of the pieces depends on the type of sequencing you are trying to do and the purpose of the sequencing. In our case, we want pieces averaging 500 base pairs that are at maximum 800 base pairs. [ii]
2. To add adapters to each piece.

The differences in library preparation methods are largely differences in the mechanisms by which these two goals are accomplished. For example, the DNA can be chopped enzymatically or mechanically or the adapters can be added by one or a number of enzymatic steps.

Pros and Cons of Library Preparation Methods:

Each step of each preparation method has various advantages and disadvantages associated with it. The primary factors for concern in library preparation are:

•  Amount of genomic DNA required – in general, the more steps involved in a preparation technique, the more genomic DNA will be required because some DNA will be lost at each step.
• Cutting bias – certain cutting techniques may be biased depending on the DNA sequence. This generally more of a concern in enzymatic cutting than in mechanical cutting.
• G-C content – Amplification steps (i.e. PCR in a thermocycler) tend to change the average G-C content of the DNA sample by favorable amplifying sequences based on the amount of guanine and cytosine in them. In general, using fewer amplification steps will decrease this bias. [iii]
• Price – the preparation methods vary widely in price, this can be a limiting factor.

 Our Methods:

For our libraries we will be using sonication (sound) to chop up the genomic DNA followed by a series of enzyme treatments from the Illumina library preparation kit that will first prepare the DNA pieces for annealing the adapters and then carry our the annealing process itself.

The adapters we are using will each contain a “barcode,” a short sequence of bases unique to each sample. Barcoding allows us to pool our samples and run them on a single Illumina well bringing down the cost of sequencing significantly.

Once we have the sequences back, we will begin the computationally challenging process of assembling and annotating them.

Our genomic library preparation kit finally came in (about a week late) but neither of the mentors are around to explain how to use it, so I thought I’d do some research and see what I could find on the Microbacterium genus. Unfortunately, most Google results are for the Mycobacterium genus which, while a very fascinating genus I’m sure, does not help me at all.

I finally came across a study performed in the mid 1960’s which analyzed some characteristics of the Microbacterium genus. The term Microbacterium was proposed in the early 20th century to identify a particular group of very small, gram-positive, rod-shaped bacteria that have been found in many dairy products. An important characteristic of Microbacterium species is that they are unusually heat resistant. The researchers conducted a number of tests to learn more about the physiological characteristics of the genus, based on 25 unique strains.

Particularly of interest to me is the following:

• These bacteria grow aerobically, but some strains can grow under anaerobic conditions.  However, they divide much more slowly and lack pigmentation when grown anaerobically. Each strain in the study grew at a decent rate (typically around 3 days of incubation, but as much as 7 days were needed in some cases) when incubated in the 30-37 degree C range. Only 5 strains grew at 39 degrees and only 3 strains grew at 9 degrees.
• All strains grew in mediums with a pH of 6.8 and 7.5 . More acidic conditions (pH < 6) yielded almost no growth from the strains.
• Some members of the genus have the ability to reduce nitrates and liquefy casein, while nearly every strain could hydrolyze gelatin
• 5 strains were able to withstand 85 degree heat, and every strain was able to withstand at least 60 degree heat
• There is doubt as t0 where this genus should be placed in a phylogenetic tree. At the time of publication of this study, the most recent proposal was to place Microbacterium within the Corynebacterium. The author, however, dismissed this based on what he observed in terms of heat resistance and ability to be cultured, noting large differences between the two.

Here is a link to the paper