developmental stage, sex, environmental conditions, or experimental challenge than those individuals you will include in your
proteomic analysis. You can get access to similar information if
you look at BioProject database (https://www.ncbi.nlm.nih.
gov/bioproject) where a collection of biological data related
to a single initiative, organization, or consortium including
links to different data sets can be found. Information about
descriptions of biological source material and experimental
details related to these projects can be found also in BioSample
database (https://www.ncbi.nlm.nih.gov/biosample). Once
you have downloaded raw data files following the NCBI guidelines, it would be necessary to complete the assembly of raw
reads using appropriate software in order to generate a consensus transcriptome (for nonexpert users OmicsBox: https://
www.biobam.com/omicsbox could be a good user-friendly
solution though this is not a freeware; if you want to use
freeware, Galaxy platform could be a very good choice to do
this job: https://usegalaxy.org). Once the reference transcriptome database is ready in FASTA format, you would need to
produce the corresponding protein database following the
steps described in the previous paragraph. Some bioinformatic
tools have been developed to elaborate customized protein
databases from RNA-seq data, for instance, customProDB
[31], though this might not be a straightforward option for
nonexpert users. If you are lucky enough, either you can find
and download assembled transcriptomic sequence databases in
FASTA format associated to BioProjects from Transcriptome
Shotgun Assembly (TSA) sequence database (searches to look
for specific organisms can be easily carried out using the different filters available in https://www.ncbi.nlm.nih.gov/Traces/
wgs/?term¼tsa) or similar FASTA files (as well as raw files in
the SRA database) containing nucleotide sequences from a
consensus transcriptome obtained in different projects published as supplementary material or in journals that are specifically dedicated to publishing big data sets like these. For
example, if you work with marine mussels, you can get this
type of FASTA file in Moreira et al. [32] and Diz et al. [33].
4. If previous steps are not good options in your case, then you
would need to think seriously in running a high-throughput
transcriptomic study in parallel to proteomic analysis [7, 27],
which would provide very useful complementary information
about the biological question under study.
5. In addition, de novo interpretation of the MS/MS spectra, a
very useful tool especially when working with non-model
organisms, can provide complementary results when combined
with the use of custom protein databases, for example, reporting possible mutations or errors in sequencing. An added value
Shotgun Proteomics in Non-model Organisms
91
proteomic analysis. You can get access to similar information if
you look at BioProject database (https://www.ncbi.nlm.nih.
gov/bioproject) where a collection of biological data related
to a single initiative, organization, or consortium including
links to different data sets can be found. Information about
descriptions of biological source material and experimental
details related to these projects can be found also in BioSample
database (https://www.ncbi.nlm.nih.gov/biosample). Once
you have downloaded raw data files following the NCBI guidelines, it would be necessary to complete the assembly of raw
reads using appropriate software in order to generate a consensus transcriptome (for nonexpert users OmicsBox: https://
www.biobam.com/omicsbox could be a good user-friendly
solution though this is not a freeware; if you want to use
freeware, Galaxy platform could be a very good choice to do
this job: https://usegalaxy.org). Once the reference transcriptome database is ready in FASTA format, you would need to
produce the corresponding protein database following the
steps described in the previous paragraph. Some bioinformatic
tools have been developed to elaborate customized protein
databases from RNA-seq data, for instance, customProDB
[31], though this might not be a straightforward option for
nonexpert users. If you are lucky enough, either you can find
and download assembled transcriptomic sequence databases in
FASTA format associated to BioProjects from Transcriptome
Shotgun Assembly (TSA) sequence database (searches to look
for specific organisms can be easily carried out using the different filters available in https://www.ncbi.nlm.nih.gov/Traces/
wgs/?term¼tsa) or similar FASTA files (as well as raw files in
the SRA database) containing nucleotide sequences from a
consensus transcriptome obtained in different projects published as supplementary material or in journals that are specifically dedicated to publishing big data sets like these. For
example, if you work with marine mussels, you can get this
type of FASTA file in Moreira et al. [32] and Diz et al. [33].
4. If previous steps are not good options in your case, then you
would need to think seriously in running a high-throughput
transcriptomic study in parallel to proteomic analysis [7, 27],
which would provide very useful complementary information
about the biological question under study.
5. In addition, de novo interpretation of the MS/MS spectra, a
very useful tool especially when working with non-model
organisms, can provide complementary results when combined
with the use of custom protein databases, for example, reporting possible mutations or errors in sequencing. An added value
Shotgun Proteomics in Non-model Organisms
91