Tuesday notes

MtGIRAFFE

Continuing work on script. Treating data as diploid after finding 24 heterozygous SNPs in VCF data.

Completed v1.0.0 of analysis at commit abeaf0eec8!

ApTranscriptome

Debugging Sailfish bootstrap on EC2…

Silly mistake left from debugging caused results to rewrite each other. Fixed and started 20 reps.

ApPopGen

Using Sabre to demultiplex data. Need to make barcode.txt file in this format

AGTGAGAAGCAGTGGTATCAACGCAGAGT   sabreN_1.fq
TACGGAAAGCAGTGGTATCAACGCAGAGT   sabreC_1.fq

where the first column is the barcode and the second is the outfile name.

Generated dd_RAD_barcodes_1-48.txt"

ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCATGCATG  GCATG_flex.fastq
ACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCACATG  AACCA_flex.fastq
ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGATCCATG  CGATC_flex.fastq
ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCGATCATG  TCGAT_flex.fastq
...

Command to allow 5 mismatches in barcode:

sabre se -m 5 -f scahan_VGN_20140730_2014_ddRad1_R1.fastq -b ddRAD_barcodes_1-48.txt -u unknown_barcodes.fastq &

Results

Total FastQ records: 152946005

FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTACCCATG: 108
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGGAACATG: 123
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCTGCCATG: 54
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCGGCATG: 5
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAGTCATG: 1
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCACGCATG: 7
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTTATACCATG: 77
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTTAGTACATG: 35
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTTACGTCATG: 0
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTTACCGCATG: 55
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTCGACATG: 2
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTCCGCATG: 4
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTAGTCATG: 0
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCTCCATG: 91
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGCCACATG: 0
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGATACATG: 0
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCTGACATG: 0
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCGTCATG: 0
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTGAGTCCATG: 0
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTGAGATCATG: 0
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTGACACCATG: 1
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGGCATG: 1
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGTCCATG: 1
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGCGCATG: 59
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGATCATG: 0
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTCGCATG: 0
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGTACCATG: 41
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGGTACATG: 2
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGGCTCATG: 5
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGAATCATG: 0
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTCATATCATG: 4
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTATTACCATG: 30
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTATGAGCATG: 19
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTATACGCATG: 20
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTACTTCCATG: 1
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTACTGGCATG: 0
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTACGGTCATG: 1
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTAATTACATG: 1
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTACACACATG: 7
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCTACATG: 35
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGGACATG: 2
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGTTGCATG: 2
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTCAACCCATG: 14
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTTGCATCATG: 10
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCGATCATG: 1
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGATCCATG: 3
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCACATG: 24
FastQ records for barcode ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCATGCATG: 4

FastQ records with no barcode match: 152945155

Number of mismatches allowed: 5

Definitely not good. Turns out that I should only have used the barcode sequence (positions 34-38) for demultiplexing. Also worth noting that the barcodes are still unique if the first posiiton is missing, as many of the reads have an N at the first position.

GCATG   GCATG_flex.fastq
AACCA   AACCA_flex.fastq
CGATC   CGATC_flex.fastq

Re-ran sabre allowing single mismatch (-m) to deal with N at first position. Much better results!

FastQ records for barcode TTACC: 4275731
FastQ records for barcode TGGAA: 2595394
FastQ records for barcode TCTGC: 2552441
FastQ records for barcode TCCGG: 3196900
FastQ records for barcode TCAGT: 2493173
FastQ records for barcode TCACG: 3381451
FastQ records for barcode TATAC: 3288965
FastQ records for barcode TAGTA: 2751938
FastQ records for barcode TACGT: 5171345
FastQ records for barcode TACCG: 5537585
FastQ records for barcode GTCGA: 5046445
FastQ records for barcode GTCCG: 4767314
FastQ records for barcode GTAGT: 1975614
FastQ records for barcode GGCTC: 2935593
FastQ records for barcode GGCCA: 2541945
FastQ records for barcode GGATA: 1575601
FastQ records for barcode GCTGA: 2368128
FastQ records for barcode GCCGT: 2359235
FastQ records for barcode GAGTC: 3006513
FastQ records for barcode GAGAT: 2067871
FastQ records for barcode GACAC: 4726443
FastQ records for barcode CTTGG: 2579598
FastQ records for barcode CTGTC: 1085679
FastQ records for barcode CTGCG: 3175215
FastQ records for barcode CTGAT: 2544480
FastQ records for barcode CGTCG: 3605274
FastQ records for barcode CGTAC: 2035376
FastQ records for barcode CGGTA: 288454
FastQ records for barcode CGGCT: 2023708
FastQ records for barcode CGAAT: 2584915
FastQ records for barcode CATAT: 7331024
FastQ records for barcode ATTAC: 2586808
FastQ records for barcode ATGAG: 4612287
FastQ records for barcode ATACG: 4287136
FastQ records for barcode ACTTC: 1518915
FastQ records for barcode ACTGG: 1874829
FastQ records for barcode ACGGT: 2962749
FastQ records for barcode AATTA: 2276665
FastQ records for barcode ACACA: 2899588
FastQ records for barcode AGCTA: 2639168
FastQ records for barcode AAGGA: 1281569
FastQ records for barcode GGTTG: 2858257
FastQ records for barcode CAACC: 6181619
FastQ records for barcode TGCAT: 2792088
FastQ records for barcode TCGAT: 1083199
FastQ records for barcode CGATC: 5194011
FastQ records for barcode AACCA: 5852849
FastQ records for barcode GCATG: 5579773

FastQ records with no barcode match: 595147

Number of mismatches allowed: 1

Next, used sickle for quality-trimmming. Sample AGCTA is middle-of-the-road, so used this as trial.

sickle se -f AGCTA_flex.fastq -t sanger -o CAACC_flex_trimmed.fastq -q 20 -l 20

Most reads retained!

Instead of fastqc, looking into using qrqc as it’s implemented in R and therefore more extensible (by me).