...
| Table of Contents |
|---|
Introduction
High-throughput
...
sequencing
...
of
...
cDNA,
...
also
...
called
...
RNA-Seq,
...
can
...
provide
...
many
...
levels
...
of
...
information
...
about
...
gene
...
expression,
...
such
...
as
...
information
...
about
...
previously
...
unannotated
...
genes,
...
expression
...
of
...
pseudogenes,
...
differential
...
expression
...
both
...
within
...
and
...
across
...
samples,
...
and
...
alternative
...
splicing.
...
Making
...
the
...
libraries
...
and
...
sending
...
them
...
out
...
for
...
sequencing
...
is
...
only
...
the
...
first
...
step
...
in
...
performing
...
an
...
RNA-Seq
...
experiment.
...
What
...
most
...
people
...
find
...
is
...
that
...
processing,
...
analyzing,
...
and
...
interpreting
...
the
...
data
...
can
...
often
...
be
...
just
...
as
...
time-consuming.
...
Take
...
heart.
...
These
...
data
...
are
...
sets
...
are
...
so
...
rich
...
that
...
you
...
may
...
never
...
fully
...
exhaust
...
their
...
potential.
...
However,
...
there
...
are
...
a
...
few
...
first
...
steps
...
you'll
...
want
...
to
...
perform
...
right
...
away,
...
as
...
well
...
as
...
some
...
quality
...
control
...
steps
...
that
...
will
...
help
...
you
...
assess
...
how
...
well
...
your
...
experiment
...
worked.
...
What
...
follows
...
is
...
a
...
description
...
of
...
RNA-Seq
...
processing
...
steps
...
we
...
do
...
fairly
...
routinely
...
in
...
the
...
Loraine
...
lab,
...
along
...
with
...
links
...
to
...
software
...
programs
...
we've
...
developed
...
for
...
in-house
...
use.
...
Please
...
be
...
aware
...
that
...
these
...
programs
...
are
...
very
...
much
...
works
...
in
...
progress
...
and
...
so
...
may
...
not
...
always
...
work
...
as
...
advertised.
...
If
...
you
...
find
...
bugs
...
or
...
inconsistencies,
...
please
...
let
...
us
...
know
...
-
...
contact
...
Ann
...
(aloraine@uncc.edu)
...
with
...
feedback,
...
suggestions,
...
and
...
bug
...
reports.
...
RNA-Seq
...
tutorial
...
The
...
following
...
protocol
...
describes
...
processing
...
data
...
from
...
Illumina
...
HiSeq
...
pipeline.
...
Whether
...
you
...
should
...
perform
...
exactly
...
these
...
steps
...
with
...
your
...
data
...
sets
...
will
...
depend
...
on
...
your
...
data
...
-
...
its
...
age,
...
whether
...
you've
...
done
...
single-end
...
or
...
paired-end
...
sequencing,
...
the
...
read
...
lengths,
...
your
...
reference
...
genome,
...
and
...
so
...
on.
...
For
...
example,
...
when
...
you
...
run
...
TopHat,
...
you
...
may
...
need
...
to
...
adjust
...
parameters
...
to
...
accommodate
...
smaller
...
read
...
lengths
...
if
...
you
...
are
...
running
...
data
...
from
...
pre-HiSeq
...
instruments.
...
Check
...
sequence
...
quality.
...
Your
...
first
...
step
...
upon
...
downloading
...
your
...
reads
...
will
...
be
...
to
...
check
...
the
...
quality
...
of
...
your
...
sequence.
...
One
...
of
...
the
...
easiest
...
to
...
use
...
tools
...
we
...
(in
...
the
...
Loraine
...
lab)
...
have
...
found
...
for
...
quality
...
checking
...
is
...
a
...
terrific
...
program
...
called
...
...
,
...
from
...
the
...
Babraham
...
Institute.
...
You
...
can
...
run
...
it
...
interactively
...
or
...
as
...
a
...
command
...
line
...
tool,
...
and
...
it's
...
written
...
in
...
Java,
...
which
...
means
...
you
...
can
...
run
...
it
...
on
...
a
...
Mac,
...
a
...
Linux
...
machine,
...
or
...
a
...
Windows
...
computer.
...
Like
...
IGB,
...
any
...
computer
...
that
...
supports
...
Java
...
can
...
run
...
FastQC.
...
What
...
you
...
should
...
be
...
looking
...
for
...
in
...
your
...
data
...
is
...
evidence
...
of
...
poor
...
sequencing
...
quality
...
as
...
well
...
as
...
spots
...
in
...
your
...
sequence
...
where
...
you
...
have
...
lots
...
and
...
lots
...
of
...
N
...
characters
...
-
...
these
...
usually
...
correspond
...
to
...
place
...
with
...
poor
...
quality.
...
If
...
your
...
reads
...
are
...
generally
...
low
...
quality,
...
you
...
should
...
ask
...
your
...
sequencing
...
facility
...
to
...
try
...
again.
...
If
...
your
...
data
...
have
...
many,
...
many
...
over-represented
...
sequences
...
(something
...
FastQC
...
can
...
tell
...
you),
...
then
...
you
...
may
...
want
...
to
...
make
...
another
...
library
...
for
...
that
...
sample.
...
In
...
our
...
experience,
...
low
...
yields
...
and
...
low
...
quality
...
generally
...
happen
...
because
...
something
...
went
...
wrong
...
with
...
sequence.
...
Over-represented
...
sequences
...
and
...
so-called
...
PCR
...
duplicates
...
(many
...
copies
...
of
...
the
...
same
...
sequence)
...
are
...
usually
...
due
...
to
...
problems
...
in
...
library
...
construction.
...
Align your sequence.
Let's
...
assume
...
that
...
(happily)
...
you
...
have
...
good-quality
...
sequence.
...
Your
...
next
...
step
...
should
...
be
...
to
...
align
...
your
...
sequences
...
onto
...
a
...
reference
...
genome
...
or
...
refernece
...
transcriptome.
...
Indeed,
...
even
...
if
...
you
...
haven't
...
got
...
high-quality
...
sequence,
...
you
...
should
...
still
...
try
...
to
...
align
...
it,
...
because
...
the
...
alignments
...
can
...
tell
...
you
...
a
...
lot
...
about
...
what
...
went
...
wrong.
...
For
...
this
...
tutorial,
...
we'll
...
use
...
a
...
spliced
...
alignment
...
tool
...
to
...
align
...
the
...
RNA-Seq
...
reads
...
onto
...
a
...
reference
...
genome.
...
For RNA-Seq
...
data
...
sets,
...
we
...
mainly
...
have
...
used
...
TopHat,
...
from
...
the
...
University
...
of
...
Maryland.
...
Others
...
are
...
available,
...
but
...
since
...
we
...
have
...
the
...
most
...
experience
...
with
...
TopHat
...
and
...
seems
...
to
...
be
...
one
...
of
...
the
...
more
...
widely
...
used
...
programs,
...
the
...
following
...
examples
...
will
...
demonstrate
...
how
...
to
...
run
...
it.
...
TopHat
...
is
...
a
...
spliced
...
alignment
...
tool
...
that
...
first
...
runs
...
BowTie
...
(a
...
non-spliced
...
alignment
...
tool
...
from
...
the
...
same
...
group)
...
and
...
then
...
attempts
...
to
...
align
...
any
...
reads
...
BowTie
...
couldn't
...
align
...
by
...
splitting
...
them
...
across
...
putative
...
introns.
...
For
...
this
...
reason,
...
to
...
run
...
TopHat
...
you'll
...
have
...
to
...
install
...
BowTie.
...
You'll
...
also
...
have
...
to
...
install
...
samtools,
...
a
...
program
...
that
...
TopHat
...
and
...
BowTie
...
use
...
to
...
generate
...
alignment
...
files
...
called
...
"BAM"
...
(binary
...
alignment)
...
files.
...
IGB
...
can
...
display
...
data
...
from
...
BAM
...
files,
...
once
...
you've
...
created
...
an
...
index
...
for
...
them.
...
More
...
on
...
indexing
...
BAM
...
files
...
will
...
come
...
later.
...
Many
...
different
...
versions
...
of
...
TopHat
...
have
...
been
...
released
...
over
...
the
...
past
...
couple
...
of
...
years
...
and
...
each
...
behaves
...
slightly
...
differently.
...
However,
...
a
...
few
...
things
...
seem
...
to
...
remain
...
stable.
...
First,
...
TopHat
...
will
...
typically
...
report
...
multiple
...
alignments
...
for
...
some
...
number
...
of
...
reads.
...
This
...
is
...
to
...
be
...
expected.
...
However,
...
depending
...
on
...
your
...
experimental
...
goals,
...
you
...
may
...
want
...
to
...
focus
...
on
...
the
...
reads
...
that
...
map
...
exactly
...
once
...
onto
...
the
...
genome.
...
You
...
can
...
figure
...
out
...
which
...
reads
...
mapped
...
to
...
multiple
...
locations
...
by
...
looking
...
at
...
the
...
"NH"
...
flag
...
in
...
each
...
alignment.
...
Also,
...
you
...
should
...
determine
...
the
...
minimum
...
and
...
maximum
...
intron
...
sizes
...
for
...
your
...
genome
...
and
...
provide
...
these
...
as
...
parameters
...
to
...
TopHat.
...
For
...
details
...
on
...
running
...
TopHat,
...
see
...
the
...
...
...
.
Here is an example invocation of TopHat, fine-tuned
...
for
...
Arabidopsis
...
thaliana
...
.
...
TopHat
...
is
...
a
...
command
...
line
...
program,
...
which
...
means
...
you
...
run
...
it
...
by
...
typing
...
commands
...
into
...
a
...
Unix
...
terminal.
...
(On
...
Mac,
...
this
...
is
...
the
...
Terminal
...
program
...
from
...
the
...
Applications
...
Utilities.)
...
For
...
the
...
rest
...
of
...
this
...
tutorial,
...
wherever
...
you
...
see
...
a
...
line
...
that
...
starts
...
with
...
a
...
"$"
...
sign,
...
this
...
means
...
you
...
type
...
the
...
command
...
into
...
a
...
terminal.
...
(The
...
"$"
...
means:
...
the
...
Unix
...
prompt.)
...
| Code Block |
|---|
$ tophattophat2 --max-intron-length 2000 /data/bowtieindex/A_thaliana_Jun_2009 Sample.fastq -o Sample {code} |
However,
...
you're
...
probably
...
better
...
off
...
running
...
TopHat
...
on
...
a
...
fairly
...
powerful
...
machine.
...
If
...
you
...
can
...
find
...
a
...
multi-processor
...
server
...
with
...
16
...
or
...
more
...
of
...
RAM,
...
I
...
would
...
strongly
...
recommend
...
using
...
it
...
to
...
run
...
the
...
alignment
...
step.
...
If
...
you
...
do
...
get
...
access
...
to
...
a
...
multi-processor
...
server,
...
you
...
should
...
tell
...
TopHat
...
to
...
take
...
advantage
...
of
...
the
...
extra
...
processing
...
power
...
using
...
the
...
-p
...
parameter
...
that
...
allows
...
you
...
to
...
specify
...
the
...
number
...
of
...
processors
...
TopHat
...
can
...
use.
...
To
...
run
...
bowtie2,
...
you
...
have
...
to
...
first
...
make
...
an
...
index
...
file
...
for
...
your
...
genome.
...
And
...
to
...
make
...
an
...
index
...
for
...
your
...
genome,
...
you'll
...
need
...
a
...
fasta
...
file
...
for
...
your
...
genome.
...
This
...
is
...
easy
...
to
...
get,
...
however.
...
You
...
can
...
either
...
download
...
the
...
fasta
...
files
...
directly
...
from
...
the
...
genome
...
data
...
provider
...
(e.g.,
...
UCSC
...
or
...
NBCI)
...
or
...
you
...
can
...
get
...
a
...
2bit
...
file
...
from
...
the
...
IGBQuickLoad
...
Web
...
site
...
and
...
convert
...
it
...
to
...
fasta.
...
To
...
get
...
a
...
sequence
...
data
...
file
...
from
...
IGBQuickLoad.org,
...
go
...
to
...
the
...
genome
...
directory
...
and
...
download
...
the
...
"2bit"
...
file
...
you
...
find
...
there.
...
Then
...
use
...
Jim
...
Kent's
...
2bit2
...
In
...
this
...
example,
...
I'll
...
run
...
bowtie2-build
...
using
...
a
...
fasta
...
file
...
A_thaliana_Jun_2009.fa
...
to
...
create
...
index
...
files
...
bowtie2 uses
...
to
...
speed
...
up
...
the
...
alignment
...
process.
...
We
...
TopHat
...
will
...
created
...
a
...
directory
...
called
...
Sample
...
(the
...
-o
...
parameter).
...
In
...
IGB,
...
we
...
use
...
files
...
in
...
"2bit"
...
format
...
to
...
represent
...
genomic
...
sequence
...
data.
...
It's
...
easy
...
to
...
convert
...
a
...
2bit
...
file
...
into
...
a
...
fasta
...
file
...
and
...
back
...
again.
...
To
...
create
...
a
...
fasta
...
file,
...
do something like:
| Code Block |
|---|
twoBitToFa A_thaliana_Jun_2009.2bit A_thaliana_Jun_2009.fa
|
Please note that you need to make sure that the names of chromosomes in the fasta file (and hence the BowTie index files) match with what IGB uses. For Arabidopsis, chromosome names are chr1, chr2, chr3, chr4, chr5, chrC, and chrM.
IGB has a synonyms system that allows it to "understand" that chr1 and Chr1 are really the same thing, but other programs you will run into might not be able to match names in this way. For this reason, it's a good idea to use the same names for chromosome throughout all the different steps of processing data.
Process output files
Index (and rename) your alignment file
When TopHat finishes, it will have created a file called "accepted_hits.bam"
...
that
...
contains
...
your
...
alignments.
...
Your
...
next
...
step
...
in
...
the
...
process
...
will
...
be
...
to
...
index
...
this
...
file,
...
creating
...
what's
...
called
...
a
...
"bai"
...
index
...
file.
...
For
...
this,
...
you'll
...
use
...
samtools,
...
a
...
freely
...
available
...
command-line
...
tool
...
with
...
many
...
useful
...
functions.
...
You
...
can
...
learn
...
about
...
samtools
...
at
...
...
...
...
and
...
download
...
the
...
code
...
from
...
...
...
hosted
...
at
...
sourceforge.net.
...
Why
...
make
...
an
...
index?
...
This
...
is
...
a
...
crucial
...
step
...
because
...
it
...
allows
...
programs
...
like
...
IGB
...
to
...
do
...
what
...
is
...
called
...
a
...
"region-based
...
query."
...
That
...
is,
...
once
...
you've
...
started
...
IGB,
...
you
...
can
...
load
...
the
...
BAM
...
file,
...
zoom
...
in
...
on
...
a
...
region,
...
and
...
then
...
ask
...
IGB
...
to
...
load
...
in
...
just
...
the
...
reads
...
that
...
overlap
...
your
...
region
...
of
...
interest.
...
This
...
works
...
because
...
IGB
...
reads
...
the
...
index
...
file,
...
which
...
tells
...
IGB
...
exactly
...
where
...
to
...
look
...
in
...
the
...
larger
...
BAM
...
file
...
(which
...
may
...
be
...
1
...
gig
...
or
...
more
...
!)
...
for
...
just
...
the
...
subset
...
of
...
reads
...
that
...
are
...
relevant
...
to
...
the
...
region
...
in
...
view.
...
To
...
make
...
an
...
index
...
(.bai)
...
file
...
for
...
a
...
BAM
...
file,
...
you
...
typically
...
would
...
first
...
have
...
to
...
sort
...
the
...
BAM
...
file
...
first.
...
However,
...
the
...
"accepted_hits.bam"
...
file
...
from
...
TopHat
...
should
...
already
...
be
...
sorted.
...
So
...
all
...
you
...
should
...
need
...
to
...
do
...
next
...
is
...
rename
...
it
...
and
...
make
...
an
...
index
...
for
...
it,
...
like
...
so:
...
| Code Block |
|---|
$ mv accepted_hits.bam Sample.bam $ samtools index Sample.bam {noformat} |
which
...
will
...
create
...
an
...
index
...
file
...
named
...
Sample.bam.bai.
...
Make
...
a
...
bedgraph
...
(wiggle)
...
genome
...
coverage
...
file
...
A
...
genome
...
coverage
...
(or
...
depth)
...
file
...
reports
...
the
...
number
...
of
...
reads
...
overlapping
...
regions
...
or
...
individual
...
bases
...
of
...
the
...
reference
...
genome.
...
Older
...
versions
...
of
...
TopHat
...
used
...
to
...
create
...
a
...
coverage
...
file,
...
but
...
more
...
recent
...
versions
...
of
...
Tophat
...
no
...
longer
...
do
...
this.
...
However,
...
you
...
can
...
create
...
coverage
...
graphs
...
using
...
bedtools
...
...
command.
...
For
...
information
...
on
...
where
...
to
...
get
...
bedtools
...
and
...
how
...
to
...
install
...
it,
...
visit
...
the
...
To create a genome coverage bedgraph file from your BAM alignments file on a Linux machine, do this:
| Code Block |
|---|
$ genomecov -ibam Sample.bam -split -bg > Sample.bedgraph {code} |
What
...
should
...
happen
...
next
...
is
...
that
...
a
...
new
...
file
...
should
...
appear
...
named
...
Sample.bedgraph,
...
which will have a structure that looks a bit like:
| Code Block |
|---|
chr1 3667 3697 2
chr1 3697 3707 3
chr1 3707 3726 5
chr1 3726 3742 6
chr1 3742 3744 4
chr1 3744 3749 5
chr1 3749 3755 6
|
At this point, you should be able to open the "bedgraph" file directly in IGB. However, IGB will work better if you sort the file, compress it using bgzip, and then make an index for the file using tabix.
| Code Block |
|---|
$ sort -k1,1 -k2,2n Sample.bedgraph | bgzip > Sample.bedgraph.gz
$ tabix -s 1 -b 2 -e 3 Sample.bedgraph.gz
{code}
|
The
...
sort
...
command
...
will
...
read
...
the
...
entire
...
Sample.bedgraph
...
file
...
into
...
memory,
...
sort
...
the
...
data,
...
and
...
then
...
output
...
the
...
data
...
in
...
sorted
...
order.
...
The
...
sorted
...
data
...
will
...
first
...
be
...
ordered
...
by
...
chromosome
...
name
...
(field
...
1)
...
and
...
then
...
by
...
interval
...
start
...
position
...
(field
...
2).
...
In
...
the
...
command
...
above,
...
the
...
sorted
...
data
...
are
...
piped
...
into
...
the
...
bgzip
...
compression
...
tool
...
and
...
then
...
the
...
compressed,
...
sorted
...
data
...
are
...
saved
...
to
...
a
...
file
...
called
...
Sample.bedgraph.gz
...
.
...
The
...
tabix
...
command
...
then
...
creates
...
an
...
index
...
file
...
(extension
...
.tbi
...
)
...
for
...
the
...
newly
...
compressed,
...
sorted
...
Sample.bedgraph.gz
...
file.
...
Note
...
:
...
To
...
view
...
Sample.bedgraph.gz
...
file
...
in
...
IGB,
...
you
...
should
...
always
...
keep
...
the
...
index
...
file
...
(named
...
Sample.bedgraph.gz.tbi
...
)
...
in
...
the
...
same
...
folder.
...
To
...
get
...
a
...
copy
...
of
...
bgzip
...
and
...
tabix
...
,
...
go
...
to
...
...
...
...
...
.
...
Note
...
that
...
development
...
of
...
tabix
...
has
...
moved
...
to
...
...
...
...
View data in IGB
Once you've
...
created
...
the
...
files,
...
you
...
should
...
then
...
be
...
able
...
to
...
open
...
them
...
in
...
IGB.
...
However,
...
be
...
sure
...
to
...
keep
...
the
...
index
...
files
...
(.bai
...
from
...
samtools
...
and
...
.tbi
...
from
...
tabix)
...
in
...
the
...
same
...
folder
...
with
...
their
...
corresponding
...
alignments
...
or
...
bedgraph
...
files.
...
Once
...
you
...
open
...
them
...
in
...
IGB,
...
scroll
...
and
...
zoom
...
to
...
a
...
smallist
...
region
...
of
...
your
...
genome
...
-
...
-
...
a
...
region
...
where
...
you
...
can
...
see
...
maybe
...
five
...
or
...
six
...
different
...
genes.
...
(Use
...
the
...
"search"
...
tab
...
to
...
zoom
...
in
...
on
...
a
...
particular
...
gene
...
of
...
interest.)
...
Then,
...
click
...
"Load
...
Data"
...
to
...
load
...
data
...
from
...
the
...
files
...
you
...
selected.
...
(All
...
selected
...
files
...
should
...
appear
...
in
...
the
...
Data
...
Management
...
Table
...
on
...
the
...
right
...
side
...
of
...
the
...
Data
...
Access
...
tab.)
...
If
...
all
...
goes
...
well,
...
the
...
bedgraph
...
file
...
data
...
should
...
appear
...
in
...
a
...
graph
...
track.
...
To
...
change
...
its
...
appearance,
...
e.g.,
...
add
...
a
...
y-axis,
...
change
...
the
...
color,
...
etc.
...
select
...
the
...
track
...
label
...
and
...
click
...
the
...
Graph
...
Adjuster
...
tab,
...
which
...
contains
...
various
...
controls
...
for
...
working
...
with
...
graphs.
...
The
...
reads
...
(from
...
the
...
BAM)
...
file
...
appear
...
in
...
tracks
...
above
...
and
...
below
...
the
...
axis.
...
To
...
change
...
how
...
reads
...
look,
...
choose
...
File->Preferences->Tracks
...
and
...
use
...
the
...
options
...
you
...
see
...
there
...
to
...
merge
...
all
...
the
...
reads
...
into
...
a
...
single
...
+/
...
-
...
track,
...
use
...
color
...
to
...
indicate
...
strand,
...
change
...
the
...
number
...
of
...
reads
...
that
...
are
...
shown
...
in
...
stacks,
...
and
...
so
...
on.
...
For
...
more
...
information
...
on
...
working
...
with
...
tracks
...
and
...
graphs,
...
please
...
see
...
the
...
...
...
...
.
And remember,
...
if
...
you
...
have
...
questions,
...
get
...
in
...
touch
...
with
...
us
...
or
...
use
...
Google.
...
All
...
IGB
...
tutorials
...
and
...
user
...
guide
...
materials
...
are
...
publicly
...
available
...
on
...
our
...
wiki
...
and
...
have
...
(mostly)
...
been
...
indexed
...
in
...
Google.