barcoded. The reverse adapter (RevAD) is never barcoded. The
stem-loop adapters are engineered each with a 3-base barcode
tag to allow sample multiplexing during sequencing. Barcodes
will appear at the beginning of the sequence reads (positions
1–3) and must be trimmed off prior to sample alignment. The
codes were selected as a group to balance base composition.
For this reason, libraries must be pooled prior to sequencing
such that each of the eight code sequences is present in equal
proportions. Said another way, samples can only be sequenced
in lots of 8, if multiplexing is desired. However, if you choose
to sequence only one sample per lane, libraries MUST be
constructed with the NON-barcoded adapters. If you multi-
plex, make sure to document which sample is tagged with
which barcode.- This chapter focuses on experimental design and library prepa-
ration for QS transciptomics. RNAseq generates a large
amount of data and there are a number of approaches for
processing this data, depending on your goals. The raw data
must be extracted. If barcodes are used, the sample must be
sorted by barcode, the barcode removed, and then aligned to a
genome. It is a good idea to remove the remaining sequences
aligning to the ribosomal sequences. Once the data is aligned
to a genome, the transcripts for each sample are processed and
quantified. There are many approaches at this step in sample
analysis. A good starting point to identify differentially
regulated genes for biological replicates is to use the DESeq
package (with a false discovery rate [FDR] cutoff of 0.05) and
proceed with genes showing twofold or more regulation rela-
tive to the reference condition. Data analysis to compare gene
expression between two conditions can be done with multiple
approaches; if available, the Avadis NGS software package is
recommended.
Acknowledgements
A number of people contributed to the methods described in this
chapter. Christopher Armour developed the NSR primer approach
and methods for library construction [2] and also supported the
development of this method for prokaryotic species. Sudha Chu-
gani and Yasuhiro Oda contributed to other aspects of method
development, including RNA isolation and library synthesis and
analysis. Additionally, this protocol utilizes a number of commer-
cially available kits. Many of the procedures are taken directly from
the kit protocols or handbooks.RNAseq of QS Regulons 191