SRSLY UMIs are attached to the i7 index, and require a bit of handling
to make it through bcl2fastq. This package helps guide that process.
Installation
This package can be installed with pip install srslyumi or cloned from
this github repository. It is compatible with python 2.7 and 3.X.
You must also have bcl2fastq or bcl2fastq2 installed. Because bcl2fastq is only compatible with linux operating systems, srslyumi must be run on a linux machine.
Running
The srslyumi command takes two arguments: 1) an existing run directory,
and 2) an output directory for the FASTQ and bcl2fastq reports.
The software will create modified versions of RunInfo.xml and SampleSheet.csv
in the output directory, and create and run a runbcl2fastq.sh executable to
demultiplex the data and assign the output files the appropriate name. This
executable can also be used to re-run the demultiplexing if needed.
Output
The final output will be fastqs containing the forward and reverse reads for each
library with the UMI in the read name. Note: bcl2fastq outputs the 9bp UMI with the
first 9 bp of read 2, separated by +. Only the 9 UMI bases are needed for analysis.
Illumina sequencing performs read cycles for the i7 and i5 indices in between the fragment reads. SRSLY UMIs are attached to the i7 reads, like this:
Order of read cycles
+------------------------+--------------+------------+----------------+-----------------------+
| Forward fragment read | i7 index | UMI | i5 index | Reverse fragment read |
+------------------------+--------------+------------+----------------+-----------------------+
Standard bcl2fastq processing
+------------------------+---------------------------+----------------+-----------------------+
| RunInfo.xml Read 1 | RunInfo.xml R2 | RunInfo.xml R3 | RunInfo.xml R4 |
+------------------------+---------------------------+----------------+-----------------------+
| FASTQ output R1 | FASTQ header, without UMI | FASTQ output R2 |
+------------------------+--------------------------------------------+-----------------------+
Reformatted bcl2fastq processing
+------------------------+--------------+------------+----------------+-----------------------+
| new RunInfo.xml Read 1 | R2 IsIndex | R3 | R4 IsIndex | R5 |
+------------------------+--------------+------------+----------------+-----------------------+
| fixed FASTQ output R1 | FASTQ header, with UMI | fixed FASTQ output R3 |
+------------------------+--------------------------------------------+-----------------------+
However, bcl2fastq can't insert UMIs into the fragment name in the FASTQ header unless it is part of etiher the output R1 or output R2. (Note that index reads as defined in the RunInfo.xml do not count as output reads.
So to solve this, we define a new RunInfo.xml that defines five
reads instead of four:
With standard bcl2fastq processing with the TrimUMI option, this
results in the UMI in the fragment name in the FASTQ files. However,
it has two side effects: the UMI includes both the 5bp of the UMI as
well as followed by the first 5bp of the actual read2. This should be
compatible with most UMI analysis. Additionally, the proper R2 file is
labeled as R3. Post-processing can easily rename the R3 to R2.
When you are working directory is the root of this repository, the same
directory that contains setup.py, you can run pip install -e . to
install the package in a form that lets you edit your code and run it
as a python package at the same time.
During delevopment tox, will setup testing virtual environments for
Python 2.7 and Python 3.6 and run all tests. Before code can accepted
to the main repository it must pass test on Python 2.7, 3.5, 3.6, 3.7,
and 3.8, which will run on GitHub automatically.
For quick tests in your current Python environment, run pytest,
though you may need to install the test dependencies as listed under
the tox section of pyproject.toml.
To run quick tests in your current environment, run pytest
To assess code coverage of the tests, run pytest --cov --report=html.