view real-time metrics using the Sequencing Analysis Viewer
(SAV). For the latest version of SAV, visit the SAV downloads
page on the Illumina website.
29. The output from FastQC will show flags of “Passed”, “Warn,”
or “Fail” for each module. The definitions of these flags are set
with certain assumptions that will not be met by all applications. Be careful to consider the results and not simply rely on
these flags to assess your data.
30. The options for this tool are listed on Galaxy. In most cases,
you can start with the default settings, but these may need to be
altered depending on the experimental question and the data
that are being analyzed. Increasing sensitivity of read mapping
generally increases the computation time.
31. This will require you have previously added the reference
genome in FASTA format to your Galaxy history by uploading
or otherwise.
32. In most cases, the default selections can be used to run the
DESeq2 tool.
References
1. Stark R, Grzelak M (2019) Hadfield J. RNA
sequencing: the teenage years. Nat Rev Genet
20(11):631–656. https://doi.org/10.1038/
s41576-019-0150-2
2. Wang Z, Gerstein M, Snyder M (2009)
RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10(1):57–63. https://
doi.org/10.1038/nrg2484
3. Rustad TR, Roberts DM, Liao RP, Sherman
DR (2009) Methods Mol Biol 465:13–21
4. Sims D, Sudbery I, Ilott NE, Heger A, Ponting
CP (2014) Sequencing depth and coverage:
key considerations in genomic analyses. Nat
Rev Genet 15(2):121–132. https://doi.org/
10.1038/nrg3642
5. Haas BJ, Chin M, Nusbaum C, Birren BW,
Livny J (2012) How deep is deep enough for
RNA-Seq profiling of bacterial transcriptomes?
BMC Genomics 13(1):734. https://doi.org/
10.1186/1471-2164-13-734
6. Han Y, Gao S, Muegge K, Zhang W, Zhou B
(2015) Advanced Applications of RNA
Sequencing and Challenges. Bioinform Biol
Insights 9(Suppl 1):29–46. Epub 2015/11/
27. https://doi.org/10.4137/BBI.S28991
7. Afgan E, Baker D, Batut B, van den Beek M,
Bouvier D, C ˇ ech M, Chilton J, Clements D,
Coraor N, Gru ¨ning BA, Guerler A, HillmanJackson J, Hiltemann S, Jalili V, Rasche H,
Soranzo
N,
Goecks
J,
Taylor
J,
Nekrutenko A, Blankenberg D (2018) The
galaxy platform for accessible, reproducible
and collaborative biomedical analyses: 2018
update. Nucleic Acids Res 46(W1):
W537–WW44.
https://doi.org/10.1093/
nar/gky379
8. Conesa A, Madrigal P, Tarazona S, GomezCabrero D, Cervera A, McPherson A, Szczesniak MW, Gaffney DJ, Elo LL, Zhang X,
Mortazavi A (2016) A survey of best practices
for RNA-seq data analysis. Genome Biol
17:13.
https://doi.org/10.1186/s13059016-0881-8
9. Huber W, Carey VJ, Gentleman R, Anders S,
Carlson M, Carvalho BS, Bravo HC, Davis S,
Gatto L, Girke T, Gottardo R, Hahne F, Hansen KD, Irizarry RA, Lawrence M, Love MI,
MacDonald J, Obenchain V, Oles AK,
Pages H, Reyes A, Shannon P, Smyth GK,
Tenenbaum D, Waldron L, Morgan M
(2015) Orchestrating high-throughput genomic analysis with Bioconductor. Nat Methods
12(2):115–121. Epub 2015/01/31. https://
doi.org/10.1038/nmeth.3252
10. Langmead
B
(2010;Chapter
11:Unit
11 7. Epub 2010/12/15) Aligning short
sequencing reads with Bowtie. Curr Protoc
Bioinformatics.
https://doi.org/10.1002/
0471250953.bi1107s32
11. Langmead B, Salzberg SL (2012) Fast gappedread alignment with Bowtie 2. Nat Methods 9
(4):357–359.
https://doi.org/10.1038/
nmeth.1923
RNAseq Workflow
511
(SAV). For the latest version of SAV, visit the SAV downloads
page on the Illumina website.
29. The output from FastQC will show flags of “Passed”, “Warn,”
or “Fail” for each module. The definitions of these flags are set
with certain assumptions that will not be met by all applications. Be careful to consider the results and not simply rely on
these flags to assess your data.
30. The options for this tool are listed on Galaxy. In most cases,
you can start with the default settings, but these may need to be
altered depending on the experimental question and the data
that are being analyzed. Increasing sensitivity of read mapping
generally increases the computation time.
31. This will require you have previously added the reference
genome in FASTA format to your Galaxy history by uploading
or otherwise.
32. In most cases, the default selections can be used to run the
DESeq2 tool.
References
1. Stark R, Grzelak M (2019) Hadfield J. RNA
sequencing: the teenage years. Nat Rev Genet
20(11):631–656. https://doi.org/10.1038/
s41576-019-0150-2
2. Wang Z, Gerstein M, Snyder M (2009)
RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10(1):57–63. https://
doi.org/10.1038/nrg2484
3. Rustad TR, Roberts DM, Liao RP, Sherman
DR (2009) Methods Mol Biol 465:13–21
4. Sims D, Sudbery I, Ilott NE, Heger A, Ponting
CP (2014) Sequencing depth and coverage:
key considerations in genomic analyses. Nat
Rev Genet 15(2):121–132. https://doi.org/
10.1038/nrg3642
5. Haas BJ, Chin M, Nusbaum C, Birren BW,
Livny J (2012) How deep is deep enough for
RNA-Seq profiling of bacterial transcriptomes?
BMC Genomics 13(1):734. https://doi.org/
10.1186/1471-2164-13-734
6. Han Y, Gao S, Muegge K, Zhang W, Zhou B
(2015) Advanced Applications of RNA
Sequencing and Challenges. Bioinform Biol
Insights 9(Suppl 1):29–46. Epub 2015/11/
27. https://doi.org/10.4137/BBI.S28991
7. Afgan E, Baker D, Batut B, van den Beek M,
Bouvier D, C ˇ ech M, Chilton J, Clements D,
Coraor N, Gru ¨ning BA, Guerler A, HillmanJackson J, Hiltemann S, Jalili V, Rasche H,
Soranzo
N,
Goecks
J,
Taylor
J,
Nekrutenko A, Blankenberg D (2018) The
galaxy platform for accessible, reproducible
and collaborative biomedical analyses: 2018
update. Nucleic Acids Res 46(W1):
W537–WW44.
https://doi.org/10.1093/
nar/gky379
8. Conesa A, Madrigal P, Tarazona S, GomezCabrero D, Cervera A, McPherson A, Szczesniak MW, Gaffney DJ, Elo LL, Zhang X,
Mortazavi A (2016) A survey of best practices
for RNA-seq data analysis. Genome Biol
17:13.
https://doi.org/10.1186/s13059016-0881-8
9. Huber W, Carey VJ, Gentleman R, Anders S,
Carlson M, Carvalho BS, Bravo HC, Davis S,
Gatto L, Girke T, Gottardo R, Hahne F, Hansen KD, Irizarry RA, Lawrence M, Love MI,
MacDonald J, Obenchain V, Oles AK,
Pages H, Reyes A, Shannon P, Smyth GK,
Tenenbaum D, Waldron L, Morgan M
(2015) Orchestrating high-throughput genomic analysis with Bioconductor. Nat Methods
12(2):115–121. Epub 2015/01/31. https://
doi.org/10.1038/nmeth.3252
10. Langmead
B
(2010;Chapter
11:Unit
11 7. Epub 2010/12/15) Aligning short
sequencing reads with Bowtie. Curr Protoc
Bioinformatics.
https://doi.org/10.1002/
0471250953.bi1107s32
11. Langmead B, Salzberg SL (2012) Fast gappedread alignment with Bowtie 2. Nat Methods 9
(4):357–359.
https://doi.org/10.1038/
nmeth.1923
RNAseq Workflow
511