Editing BioMicroCenter:RNA HTL (section)

=USEFUL INFORMATION=
==RNA Pre-load Submissions==
:Describes samples that upon submission to the BMC can be immediately plugged into the preparatory method that has been specified in the project submission form. This option is provided to reduce library preparation costs and expedite sample processing by minimizing hands-on time by a BMC staff. The specific requirements are listed for each preparatory method in the tables above. These specifications must be met in order to qualify as a pre-load, submissions otherwise may be subject to additional charges.<br> 
:Due to the nature of a pre-load submission, the entire volume of sample submitted will be used and as such, additional services are not typically offered unless coordinated with a BMC technician. It is important to keep this in mind when submitting precious samples and the BMC recommends to save a portion of sample when possibly. To avoid unnecessary RNA degradation, all RNA pre-loads should be setup the day of submission and be coordinated with BMC staff. These pre-loads should be prepared with extreme care to avoid possible contamination and reduce freeze thaw cycles to prevent degradation of the samples. Degraded or contaminated samples can have a significant impact on library preparation and sequencing results.
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==Quadrant Layout==
:At the BioMicro Center, we organize our high-throughput projects in 384-well plates using a quadrant layout. With quadrant 1 (Q1) representing one 96-well plate, quadrant 2 (Q2) representing a second 96-well plate and so on up to Q4. We ask that plates being submitted start with Q1 and arrayed column-wise. Below are diagrams illustrating quadrant layouts.
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|'''Quadrant 1 of 384-well plate''' <br> [[IMAGE:Quadrant 1.jpg|400px]]
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==Normalization==
:Normalization is referring to bringing all samples to a uniform concentration. For library preparation, uniform input mass is necessary for proper library generation and as such, calculations for normalization should be based upon concentration in ng/&micro;L. This is in contrast to normalization for pooling of final libraries, where calculations are based upon concentration in nM since the number of molecules is important to achieve a desired read distribution when sequencing.

==Unique versus Combinatorial Dual Indexing==
:Combinatorial dual indexing is a technique that uses a set of Index 1 (denoted as i7) and Index 2(denoted as i5) barcodes that are combined in a manner such that it produces distinct i7 and i5 pairs which increases multiplexing capacity for sequencing. With combinatorial dual indexes, each i7 and i5 is shared among other samples on the same plate, typically with i5's repeating across rows and i7's down columns. ''These combinations are unique but individual indexes used are not''. However, a phenomenon known as 'index hopping' has been observed when sequencing multiplexed libraries that followed single or combinatorial indexing schemes with newer Illumina platforms utilizing ExAmp chemistry such as the NovaSeq ([[Media:Index_Hopping.pdf |Costello et al., 2018]]). This swapping of indexes causes reads to be mis-assigned and subsequently excluded from further analysis. The primary strategy employed to mitigate the effects of index hopping is through the utilization of unique dual indexes.<br>

:Unique dual indexes (UDI) are non-redundant indexes where each i5 and i7 has a distinct index sequence. As opposed to combinatorial dual indexing, an i7 and i5 index is never repeated nor shared among other samples (i.e. for a 96-well UDI index plate, there are 96 unique i7's and 96 unique i5's). Both the combinations and individual indexes used are unique, and as a result the frequency of mis-assigned reads due to index hopping is greatly reduced. The BioMicro Center recommends that UDI's always be used when sequencing on the NovaSeq. The number of libraries that can be multiplexed and sequenced on a single lane is determined by the total number of UDI's provided for each library preparation method.<br><br>