I am new to NGS data analysis and trying to map my genome reads from Illumnia platform (paired-end reads, read length 100 and 150bp with insert length 350bp, and mate pair reads with read length 38bp and insert length 2kb and 5kb) to a reference genome (not the same specie, in the same family). I have tried to align these reads reference genome with BOWTIE, and BWA,).
No reads are mapped by BOWTIE at all with the following setting:
./bowtie REF --fr -I 320 -X 420 -q -1 GAIIx_150bp_1.fastq -2 GAIIx_150bp_2.fastq --fr -I 320 -X 420 -q -1 HiSeq_100bp_1.fastq -2 HiSeq_100bp_2.fastq --ff -I 1300 -X 3500 -q -1 HiSeq_s_1_1_QuaControled.fastq -2 HiSeq_s_1_2_QuaControled.fastq --ff -I 3300 -X 7600 -q -1 HiSeq_s_2_1_QuaControled.fastq -2 HiSeq_s_2_2_QuaControled.fastq -k 1 -m 1 -v 3 --al Palm_Hits_Bowtie --un Palm_NoHits_Bowtie Palm_Bowtie.sam -S --tryhard > bowtie.output
What's wrong with my setting?
I tried with BWA with one lane of 150bp reads as well using the default setting, I only got 0.26% percentage of reads being mapped with default setting, and 0.36% percentage of reads being mapped with the following command:
bwa aln -n 7 -o 3 REF.fsa GAIIx_s_1_1.fastq > GAIIx_s_1_1_bwa.sai
bwa aln -n 7 -o 3 REF.fsa GAIIx_s_1_2.fastq > GAIIx_s_1_2_bwa.sai
bwa sampe REF.fsa GAIIx_s_1_1_bwa.sai GAIIx_s_1_2_bwa.sai GAIIx_s_1_1.fastq GAIIx_s_1_2.fastq > GAIIx_s_1_bwa.sam.
All these reads have been under the following quality controls:
1. convert base quality phred(Q+64) score to phred(Q+33) score
2. Adapter trimming:
adapter sequence for read 1: GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG;
adapter sequence for read 2: ACACTCTTTCCCTACACGACGCTCTTCCGATCT;
sequence to trim from read 1: AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG
sequence to trim from read 2: AGAAAGGGATGTGCTGCGAGAAGGCTAGA minimum adapter alignment length is set as 10;
base quality score threshold set as 20;
minimum length of read after adapter sequence trimming and qualtiy score filtering is set as 120 for GAIIx 150bp reads, 70 for HiSeq 100bp reads, and 30 for HiSeq 38bp reads.
Is it possible that my quality control went wrong?
Any help will be greatly appreciated
No reads are mapped by BOWTIE at all with the following setting:
./bowtie REF --fr -I 320 -X 420 -q -1 GAIIx_150bp_1.fastq -2 GAIIx_150bp_2.fastq --fr -I 320 -X 420 -q -1 HiSeq_100bp_1.fastq -2 HiSeq_100bp_2.fastq --ff -I 1300 -X 3500 -q -1 HiSeq_s_1_1_QuaControled.fastq -2 HiSeq_s_1_2_QuaControled.fastq --ff -I 3300 -X 7600 -q -1 HiSeq_s_2_1_QuaControled.fastq -2 HiSeq_s_2_2_QuaControled.fastq -k 1 -m 1 -v 3 --al Palm_Hits_Bowtie --un Palm_NoHits_Bowtie Palm_Bowtie.sam -S --tryhard > bowtie.output
What's wrong with my setting?
I tried with BWA with one lane of 150bp reads as well using the default setting, I only got 0.26% percentage of reads being mapped with default setting, and 0.36% percentage of reads being mapped with the following command:
bwa aln -n 7 -o 3 REF.fsa GAIIx_s_1_1.fastq > GAIIx_s_1_1_bwa.sai
bwa aln -n 7 -o 3 REF.fsa GAIIx_s_1_2.fastq > GAIIx_s_1_2_bwa.sai
bwa sampe REF.fsa GAIIx_s_1_1_bwa.sai GAIIx_s_1_2_bwa.sai GAIIx_s_1_1.fastq GAIIx_s_1_2.fastq > GAIIx_s_1_bwa.sam.
All these reads have been under the following quality controls:
1. convert base quality phred(Q+64) score to phred(Q+33) score
2. Adapter trimming:
adapter sequence for read 1: GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG;
adapter sequence for read 2: ACACTCTTTCCCTACACGACGCTCTTCCGATCT;
sequence to trim from read 1: AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG
sequence to trim from read 2: AGAAAGGGATGTGCTGCGAGAAGGCTAGA minimum adapter alignment length is set as 10;
base quality score threshold set as 20;
minimum length of read after adapter sequence trimming and qualtiy score filtering is set as 120 for GAIIx 150bp reads, 70 for HiSeq 100bp reads, and 30 for HiSeq 38bp reads.
Is it possible that my quality control went wrong?
Any help will be greatly appreciated
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