Hi, all. I am currently facing a alignment problem, and I don't have any idea right now. Now, I am trying to align Rice DNase-seq to IRGSP build4 genome. I am using SRR094111.sra data, and convert it to fastq with default parameter. I use bowtie2 to do the alignment(parameter:bowtie2 -p 8 --local -x /all_format -U SRR094111.fastq -S SRR094111.sam), But I just got the following low rate alignment. I am not sure what is going on. I have tried to trim last several parameters, but I don't know how many base pair I should trim like that. Can anyone show me the detail pipeline to make right alignment? Thanks a lot.
*20896348 reads; of these: 20896348 (100.00%) were unpaired; of these: 20676176 (98.95%) aligned 0 times 152269 (0.73%) aligned exactly 1 time 67903 (0.32%) aligned >1 times*
First several line of fastq data（May helpful）:
@SRR094111.1 HWUSI-EAS465_0004:3:1:1043:13479 length=36 GGTAGTAATTGACAAAAGNTCTCGTATGCCGTCTTC +SRR094111.1 HWUSI-EAS465_0004:3:1:1043:13479 length=36 ??6;<@CCCCCC@?@<79!:59897C@CCBCBBCC# @SRR094111.2 HWUSI-EAS465_0004:3:1:1043:15713 length=36 GAATGCCTGATTGCCTGTAGGTCGTATGCCGTCTTC +SRR094111.2 HWUSI-EAS465_0004:3:1:1043:15713 length=36 CCCCCBCCCCCCCCCCCCCCCCCC?CCCCCCCCCCC @SRR094111.3 HWUSI-EAS465_0004:3:1:1043:15796 length=36 ATGGACCATCATCAGCCATCTTCGTATGCCGTCTTC +SRR094111.3 HWUSI-EAS465_0004:3:1:1043:15796 length=36 CCCCC;CC;CCCBBBCBACCCCCCAA=CCCCBCACC @SRR094111.4 HWUSI-EAS465_0004:3:1:1043:14078 length=36 TGTTACTTGACGCACAATAATTCGTATGCCGTCTTC +SRR094111.4 HWUSI-EAS465_0004:3:1:1043:14078 length=36 BAB@:BBA?BBB@B:AAA:<B?BB;B<ABBBBBBB?
The GEO description about data:
The degree of DNase I digestion was assessed by pulsed-field gel electrophoresis (PFGE: 20–60 switch time, 18 h, 6 V/cm; Bio-Rad). High molecular weight (HMW) DNA after DNase I digestion was isolated, blunt ended with T4 DNA polymerase. Biotinylated adaptor I (5’ Bio ACAGGTTCAGAGTTCTACAGTCCGAC and 5’ P- GTCG GACTGTAGAACTCTGAAC) was ligated to the DNA molecules. Dynal M-280 beads (Invitrogen) were used for enriching DNase I digested DNA ends after MmeI digestion. Adaptor II (5’ P-TCGTATGCCGTCTTCTGCTTG and 5’ CAAGCAGAAGACGGCATACGANN) was then ligated to the MmeI treated ends. The DNA sample was amplified by PCR using linker-specific primers (5’ CAAGCAGAAGACGG CATACGA and 5’AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA), and purified by PAGE for isolation of DNA fragments with about 90 bp in size. The final Illumina sequencing was performed using a primer specific to linker I (5’CCACCGACAGGTTCAGAGTTCTACAGTCCGAC).
*The following is my fastQC report with failure information, others are all good.*