area. Change gloves after moving from the postamplification
area to any other part of the lab to minimize the spreading of
amplicon.
13. Examples of fasta file names are NC_000962.fna for
M. tuberculosis or NC_008596.fna for M. smegmatis. Open
your fasta file with a text editor such as TextWrangler and
take note of the genome name on the first line, for example,
>gi|118467340|ref|NC_008596.1|. At other points in the
analysis where a file containing genome properties is required
(for example, the chromosome size file in Subheading 3.10 or a
gbk file for viewing your data on a genome browser), the
genome names must match. If there is a mismatch between
the genome names, you can simply use a text editor to edit the
name as needed.
14. You will only use read 1 to identify 5
0 ends (see Fig. 1). However, aligning with both reads may increase the accuracy of read
1 alignment, particularly if read 1 is located in a repetitive
region.
15. Note that if your goal is to study the precision of transcription
initiation, this filter should be omitted.
16. Whether or not this filter is appropriate, and the stringency
with which it is applied, depend on the goal of the experiment.
17. Note that excel may use a different line break character than
python expects. If this is an issue, use a text editor such as
TextWrangler to changer line breaks to Unix (LF).
References
1. Shell SS, Wang J, Lapierre P, Mir M, Chase MR,
Pyle MM, Gawande R, Ahmad R, Sarracino DA,
Ioerger TR, Fortune SM, Derbyshire KM, Wade
JT, Gray TA (2015) Leaderless transcripts and
small proteins are common features of the mycobacterial translational landscape. PLoS Genet 11
(11):e1005641. https://doi.org/10.1371/jour
nal.pgen.1005641
2. Martini MC, Zhou Y, Sun H, Shell SS (2019)
Defining the transcriptional and posttranscriptional landscapes of mycobacterium
smegmatis in aerobic growth and hypoxia.
Front Microbiol 10:591. https://doi.org/10.
3389/fmicb.2019.00591
3. Levin JZ, Yassour M, Adiconis X, Nusbaum C,
Thompson DA, Friedman N, Gnirke A, Regev A
(2010) Comprehensive comparative analysis of
strand-specific RNA sequencing methods. Nat
Methods 7(9):709–715. https://doi.org/10.
1038/nmeth.1491
4. Parkhomchuk D, Borodina T, Amstislavskiy V,
Banaru M, Hallen L, Krobitsch S, Lehrach H,
Soldatov A (2009) Transcriptome analysis by
strand-specific sequencing of complementary
DNA. Nucleic Acids Res 37(18):e123. https://
doi.org/10.1093/nar/gkp596
5. Vargas-Blanco DA, Zhou Y, Zamalloa LG,
Antonelli T, Shell SS (2019) mRNA degradation
rates are coupled to metabolic status in mycobacterium
smegmatis.
MBio
10(4):
e00957–e00919.
https://doi.org/10.1128/
mBio.00957-19
6. Aird D, Ross MG, Chen W-S, Danielsson M,
Fennell T, Russ C, Jaffe DB, Nusbaum C,
Gnirke A (2011) Analyzing and minimizing
PCR amplification bias in Illumina sequencing
libraries. Genome Biol 12(2):R18. https://doi.
org/10.1186/gb-2011-12-2-r18
7. Hawkins TL, O’Connor-Morin T, Roy A, Santillan C (1994) DNA purification and isolation
using a solid-phase. Nucleic Acids Res 22
(21):4543–4544
8. Rodrigue S, Materna AC, Timberlake SC, Blackburn MC, Malmstrom RR, Alm EJ, Chisholm
SW (2010) Unlocking short read sequencing for
metagenomics. PLoS One 5(7):e11840.
https://doi.org/10.1371/journal.pone.
0011840.t002
RNA 5
0
End Mapping
531
area to any other part of the lab to minimize the spreading of
amplicon.
13. Examples of fasta file names are NC_000962.fna for
M. tuberculosis or NC_008596.fna for M. smegmatis. Open
your fasta file with a text editor such as TextWrangler and
take note of the genome name on the first line, for example,
>gi|118467340|ref|NC_008596.1|. At other points in the
analysis where a file containing genome properties is required
(for example, the chromosome size file in Subheading 3.10 or a
gbk file for viewing your data on a genome browser), the
genome names must match. If there is a mismatch between
the genome names, you can simply use a text editor to edit the
name as needed.
14. You will only use read 1 to identify 5
0 ends (see Fig. 1). However, aligning with both reads may increase the accuracy of read
1 alignment, particularly if read 1 is located in a repetitive
region.
15. Note that if your goal is to study the precision of transcription
initiation, this filter should be omitted.
16. Whether or not this filter is appropriate, and the stringency
with which it is applied, depend on the goal of the experiment.
17. Note that excel may use a different line break character than
python expects. If this is an issue, use a text editor such as
TextWrangler to changer line breaks to Unix (LF).
References
1. Shell SS, Wang J, Lapierre P, Mir M, Chase MR,
Pyle MM, Gawande R, Ahmad R, Sarracino DA,
Ioerger TR, Fortune SM, Derbyshire KM, Wade
JT, Gray TA (2015) Leaderless transcripts and
small proteins are common features of the mycobacterial translational landscape. PLoS Genet 11
(11):e1005641. https://doi.org/10.1371/jour
nal.pgen.1005641
2. Martini MC, Zhou Y, Sun H, Shell SS (2019)
Defining the transcriptional and posttranscriptional landscapes of mycobacterium
smegmatis in aerobic growth and hypoxia.
Front Microbiol 10:591. https://doi.org/10.
3389/fmicb.2019.00591
3. Levin JZ, Yassour M, Adiconis X, Nusbaum C,
Thompson DA, Friedman N, Gnirke A, Regev A
(2010) Comprehensive comparative analysis of
strand-specific RNA sequencing methods. Nat
Methods 7(9):709–715. https://doi.org/10.
1038/nmeth.1491
4. Parkhomchuk D, Borodina T, Amstislavskiy V,
Banaru M, Hallen L, Krobitsch S, Lehrach H,
Soldatov A (2009) Transcriptome analysis by
strand-specific sequencing of complementary
DNA. Nucleic Acids Res 37(18):e123. https://
doi.org/10.1093/nar/gkp596
5. Vargas-Blanco DA, Zhou Y, Zamalloa LG,
Antonelli T, Shell SS (2019) mRNA degradation
rates are coupled to metabolic status in mycobacterium
smegmatis.
MBio
10(4):
e00957–e00919.
https://doi.org/10.1128/
mBio.00957-19
6. Aird D, Ross MG, Chen W-S, Danielsson M,
Fennell T, Russ C, Jaffe DB, Nusbaum C,
Gnirke A (2011) Analyzing and minimizing
PCR amplification bias in Illumina sequencing
libraries. Genome Biol 12(2):R18. https://doi.
org/10.1186/gb-2011-12-2-r18
7. Hawkins TL, O’Connor-Morin T, Roy A, Santillan C (1994) DNA purification and isolation
using a solid-phase. Nucleic Acids Res 22
(21):4543–4544
8. Rodrigue S, Materna AC, Timberlake SC, Blackburn MC, Malmstrom RR, Alm EJ, Chisholm
SW (2010) Unlocking short read sequencing for
metagenomics. PLoS One 5(7):e11840.
https://doi.org/10.1371/journal.pone.
0011840.t002
RNA 5
0
End Mapping
531