This project was completed as part of the Bioinformatics and Genomics Master's Program at the University of Oregon.
The objective was to design an algorithm to quality filter and demultiplex short-read sequencing data and assess levels of index hopping. Demultiplexing is required when sequencing libraries are pooled -- the resulting reads must be sorted by their barcodes or indexes. Index hopping can interfere with accurate downstream analyses by causing incorrect or ambiguous read classification.
The program Winans-Demultiplexer.py can be called from the command line and requires the following arguments:
-r1, --read1 FASTQ file containing read 1 ("forward reads")
-r2, --read2 FASTQ file containing read 2 ("index 1")
-r3, --read3 FASTQ file containing read 3 ("index 2")
-r4, --read4 FASTQ file containing read 4 ("reverse reads")
-i, --index_file Tab-separated file containing index data
The index file must be in the following format:
sample group treatment index index sequence
1 2A control B1 GTAGCGTA
2 2B control A5 CGATCGAT
3 2B control C1 GATCAAGG
4 2C mbnl B9 AACAGCGA
6 2D mbnl C9 TAGCCATG
7 2E fox C3 CGGTAATC
8 2F fox B3 CTCTGGAT
10 2G both C4 TACCGGAT
11 2H both A11 CTAGCTCA
14 3B control C7 CACTTCAC
15 3C mbnl B2 GCTACTCT
16 3D mbnl A1 ACGATCAG
17 3E fox B7 TATGGCAC
19 3F fox A3 TGTTCCGT
21 3G both B4 GTCCTAAG
22 3H both A12 TCGACAAG
23 4A control C10 TCTTCGAC
24 4A control A2 ATCATGCG
27 4C mbnl C2 ATCGTGGT
28 4D mbnl A10 TCGAGAGT
29 4E fox B8 TCGGATTC
31 4F fox A7 GATCTTGC
32 4G both B10 AGAGTCCA
34 4H both A8 AGGATAGC
The program output consists of six FASTQ files and a summary text file.
The output FASTQ files include:
- Forward reads with matched indexes
- Reverse reads with matched indexes
- Forward reads with hopped indexes
- Reverse reads with hopped indexes
- Forward reads with unknown or low quality indexes
- Reverse reads with unknown or low quality indexes
Example summary file:
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| Demultiplexing Summary |
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Number of matched reads: 304980270
Percent matched reads: 83.96%
Number of hopped reads: 517612
Percent hopped reads: 0.14%
Number of unknown reads: 57748853
Percent unknown reads: 15.9%
Total reads: 363246735
Matched counts per sample:
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SAMPLE INDEX SEQ COUNT PERCENT OF MATCHED READS
01 GTAGCGTA 7450201 2.44%
02 CGATCGAT 5225776 1.71%
03 GATCAAGG 6085915 2.0%
04 AACAGCGA 8178191 2.68%
06 TAGCCATG 9852258 3.23%
07 CGGTAATC 4498136 1.47%
08 CTCTGGAT 32163349 10.55%
10 TACCGGAT 69307073 22.73%
11 CTAGCTCA 16162895 5.3%
14 CACTTCAC 3833640 1.26%
15 GCTACTCT 6610857 2.17%
16 ACGATCAG 7441721 2.44%
17 TATGGCAC 10195805 3.34%
19 TGTTCCGT 14786868 4.85%
21 GTCCTAAG 8164223 2.68%
22 TCGACAAG 3548541 1.16%
23 TCTTCGAC 39149148 12.84%
24 ATCATGCG 9264615 3.04%
27 ATCGTGGT 6357656 2.08%
28 TCGAGAGT 10658212 3.49%
29 TCGGATTC 4163314 1.37%
31 GATCTTGC 3425453 1.12%
32 AGAGTCCA 10378366 3.4%
34 AGGATAGC 8078057 2.65%
Counts per hopped index pair:
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INDEX PAIR COUNT PERCENT OF HOPPED READS
('GTAGCGTA', 'CGATCGAT') 144 0.03%
('GTAGCGTA', 'GATCAAGG') 156 0.03%
('GTAGCGTA', 'AACAGCGA') 222 0.04%
('GTAGCGTA', 'TAGCCATG') 197 0.04%
('GTAGCGTA', 'CGGTAATC') 104 0.02%
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