Editing BioMicroCenter:HTLD TEST HTLD TEST (section)

==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>