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Quick Start

  1. Install Python

Launch a terminal window, and check that Python (2.7 or higher) and pip are installed.

$ python --version
$ pip --version
If Python or pip is not available, see instructions for installing here:
  1. Install Connor

This command will install connor and its dependencies; it may take a moment to start showing progress and should take several minutes to complete. See INSTALL for more info on requirements and more install options.

$ pip install connor --user
...
Successfully installed Connor-0.6.1

Run this line (and/or add it to your ~/.bashrc file) and confirm connor is in your path:

$ PATH=~/.local/bin:$PATH
$ which connor
<some_dir>/connor
$ connor --help
usage: connor input_bam output_bam
positional arguments:
...
v0.6.1

3. Download the example data:

$ git clone https://github.com/umich-brcf-bioinf/Connor sample_data
...
  1. Run Connor:

This command will read sample1-original.bam and produce sample1-deduped.bam (with BAM index) and a log file in your working directory.

$ connor sample_data/examples/sample1-original.bam sample1-deduped.bam
2016-09-12 17:08:12|INFO|connor begins
2016-09-12 17:08:12|INFO|logging to [sample1-deduped.bam.log]
2016-09-12 17:08:12|INFO|reading input bam [sample_data/examples/sample1-original.bam]
...
2016-09-12 17:08:32|INFO|6.83% (18104/264986) alignments unplaced or discarded
2016-09-12 15:08:32|INFO|families discarded: 15.21% (1950/12817) family too small (<3)
2016-09-12 15:08:32|INFO|93.17% (246882/264986) alignments included in 10867 families
2016-09-12 15:08:32|INFO|95.60% deduplication rate (1 - 10867 families/246882 included alignments)
2016-09-12 15:08:32|INFO|sorting/indexing [sample1-deduped.bam]
2016-09-12 15:08:32|INFO|connor complete  (20 seconds, 35mb peak memory)

$ ls -1 sample1*
sample1-deduped.bam
sample1-deduped.bam.bai
sample1-deduped.bam.log

From the log above, you can see that the original alignments resulted in 10867 deduplicated families (pairs).

  1. Reviewing output file

You don't need samtools installed to use Connor, but if you have samtools installed, you can examine the difference between original and deduplicated bams:

$ samtools flagstat sample_data/examples/sample1-original.bam
264986 + 0 in total (QC-passed reads + QC-failed reads)
59 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
263950 + 0 mapped (99.61%:nan%)
264927 + 0 paired in sequencing
132416 + 0 read1
132511 + 0 read2
261748 + 0 properly paired (98.80%:nan%)
262855 + 0 with itself and mate mapped
1036 + 0 singletons (0.39%:nan%)
848 + 0 with mate mapped to a different chr
834 + 0 with mate mapped to a different chr (mapQ>=5)

$ samtools flagstat sample1-deduped.bam
21734 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
21734 + 0 mapped (100.00%:nan%)
21734 + 0 paired in sequencing
10867 + 0 read1
10867 + 0 read2
21734 + 0 properly paired (100.00%:nan%)
21734 + 0 with itself and mate mapped
0 + 0 singletons (0.00%:nan%)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

Note that 264986 original alignments were deduplicated to 21734 (10867 pairs).

  1. Reviewing a consensus alignment

Connor adds a set of custom tags to each consensus alignment that provide details on the family of original alignment. Here is an excerpt of the first alignment:

$ samtools view sample1-deduped.bam | head -1 | tr '\t' '\n'
HWI-D00143:749:HM5YFBCXX:2:1112:3541:48875
99
chr3
178873584
... lines omitted ...
X1:Z:178873584~178873660
X2:Z:14S76M10S~8S76M16S
X3:i:0
X4:Z:GAAAGT~CTTCGT
X5:i:5

Note the BAM tags listed on the last few lines above (X1-X5). The documentation for these tags is in the SAM/BAM header and summarized here:

  • X1: leftmost~rightmost matched pair positions
  • X2: L~R CIGARs
  • X3: unique identifier for this alignment family
  • X4: L~R UMT barcodes for this alignment family; because of fuzzy matching the family UMT may be distinct from the UMT of the original alignment
  • X5: family size (number of align pairs in this family)

Interpreting the tag definitions with the alignment above, this consensus alignment represents 5 original alignment pairs (from tag X5 above) whose alignment leftmost and rightmost positions matched 178873584~178873660 (from tag X1) and left-right UMT barcodes matched GAAAGT~CTTCGT (from tag X4).