several currently available platforms compatible with RNAseq, as
summarized in [6]. Currently, Illumina offers three platforms:
(1) the MiSeq, which typically yields between 1 and 25 million
reads per run; (2) the NextSeq, which typically yields 130–400
million reads per run; and (3) the HiSeq, which typically yields
300 million to 4 billion per run. Multiple samples can be sequenced
within a single sequencing run by attaching a sample-specific oligonucleotide barcode to all transcripts belonging to a sample. These
barcodes can then be used during the computational postprocessing of the sequence data to determine which sets of sequenced
reads belonged to each input sample. Given that commercially
available multiplexing reagents enable the multiplexing of up to
96 unique samples, we recommend selecting the Illumina NextSeq
platform and multiplexing 40 samples per sequencing run to
achieve the desired 10 million reads per sample for RNAseq with
the goal of analyzing DGE.
Another sequencing parameter to decide is the length and
orientation of the sequenced read. Illumina platforms support
read lengths of up to 150 base pairs (NextSeq and HiSeq) or
300 base pairs (MiSeq) and support either single-end (sequencing
from either 5
0 or 3
0 end) or paired-end (sequencing both 5
0 and 3
0
ends). For RNAseq with the goal of DGE, single-end reads 50–75
base pairs in length are sufficient, whereas for de novo transcriptome assembly (i.e., for metatranscriptome profiling a sample containing an unknown set of organisms), Illumina recommends using
75 base pair paired-end sequencing.
1.2 Experimental
Component Overview
Within a typical bacterial total RNA sample, up to approximately
90% of the transcripts map to ribosomal RNA (rRNA). Given that
the goal of DGE analysis is to evaluate the expression levels of
mRNA transcripts, the relative fraction of “useful” sequence reads
that map to mRNA would be only ~10% of the total sequences
associated with a specific sample. Therefore, in the absence of rRNA
depletion (step 1), samples would necessitate substantially greater
sequence depth to achieve the same level of mRNA expression
quantitation. There are several commercially available rRNA depletion kits compatible with bacteria available, including siTOOLs
Biotech’s riboPool, Illumina’s RiboZero Plus, NEB’s rRNA depletion kit, and Thermo Fisher Scientific’s RiboMinus. The extent of
rRNA depletion achieved and the amount of output product
depend on the kit. We outline below a protocol for rRNA depletion
with riboPOOL, which we have found to achieve high rRNA
depletion in M. tuberculosis.
Once rRNA has been depleted, the RNA sample will need to
be processed into a library compatible with Illumina sequencing
(step 2). The most well-established library preparation kit for
bacteria is the NEBNext Ultra II RNA library prep kit for Illumina.
This kit enables cDNA synthesis from RNA using random primers,
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Shuyi Ma et al.
summarized in [6]. Currently, Illumina offers three platforms:
(1) the MiSeq, which typically yields between 1 and 25 million
reads per run; (2) the NextSeq, which typically yields 130–400
million reads per run; and (3) the HiSeq, which typically yields
300 million to 4 billion per run. Multiple samples can be sequenced
within a single sequencing run by attaching a sample-specific oligonucleotide barcode to all transcripts belonging to a sample. These
barcodes can then be used during the computational postprocessing of the sequence data to determine which sets of sequenced
reads belonged to each input sample. Given that commercially
available multiplexing reagents enable the multiplexing of up to
96 unique samples, we recommend selecting the Illumina NextSeq
platform and multiplexing 40 samples per sequencing run to
achieve the desired 10 million reads per sample for RNAseq with
the goal of analyzing DGE.
Another sequencing parameter to decide is the length and
orientation of the sequenced read. Illumina platforms support
read lengths of up to 150 base pairs (NextSeq and HiSeq) or
300 base pairs (MiSeq) and support either single-end (sequencing
from either 5
0 or 3
0 end) or paired-end (sequencing both 5
0 and 3
0
ends). For RNAseq with the goal of DGE, single-end reads 50–75
base pairs in length are sufficient, whereas for de novo transcriptome assembly (i.e., for metatranscriptome profiling a sample containing an unknown set of organisms), Illumina recommends using
75 base pair paired-end sequencing.
1.2 Experimental
Component Overview
Within a typical bacterial total RNA sample, up to approximately
90% of the transcripts map to ribosomal RNA (rRNA). Given that
the goal of DGE analysis is to evaluate the expression levels of
mRNA transcripts, the relative fraction of “useful” sequence reads
that map to mRNA would be only ~10% of the total sequences
associated with a specific sample. Therefore, in the absence of rRNA
depletion (step 1), samples would necessitate substantially greater
sequence depth to achieve the same level of mRNA expression
quantitation. There are several commercially available rRNA depletion kits compatible with bacteria available, including siTOOLs
Biotech’s riboPool, Illumina’s RiboZero Plus, NEB’s rRNA depletion kit, and Thermo Fisher Scientific’s RiboMinus. The extent of
rRNA depletion achieved and the amount of output product
depend on the kit. We outline below a protocol for rRNA depletion
with riboPOOL, which we have found to achieve high rRNA
depletion in M. tuberculosis.
Once rRNA has been depleted, the RNA sample will need to
be processed into a library compatible with Illumina sequencing
(step 2). The most well-established library preparation kit for
bacteria is the NEBNext Ultra II RNA library prep kit for Illumina.
This kit enables cDNA synthesis from RNA using random primers,
484
Shuyi Ma et al.