# QC & trimming ## QC The trimming process is run with 2 threads (`-t 2`) and took about **1.3** hours to complete. Results are placed in the `fastqc` folder ```bash $ cd /home/USER/SSAPs $ mkdir fastqc $ declare -a runname=("ERR2675454" "ERR2675455" "ERR2675458" "ERR2675459" "ERR2675460" "ERR2675461" "ERR2675464" "ERR2675465" "ERR2675468" "ERR2675469" "ERR2675472" "ERR2675473" "ERR2675476" "ERR2675477" "ERR2675478" "ERR2675479" "ERR2675480" "ERR2675481" "ERR2675484" "ERR2675485") for id in ${runname[@]}; do fq1=fastqs/${id}_1.fastq.gz fq2=fastqs/${id}_2.fastq.gz fastqc -t 2 --extract -o fastqc $fq1 $fq2 done ``` Results can be view by opening the `*.html` files in web browser or `summary.txt` and`fastqc_data.txt` in the output folders {% code title="fastqc/ERR2675454\_1\_fastqc/summary.txt" %} ```bash PASS Basic Statistics ERR2675454_1.fastq.gz PASS Per base sequence quality ERR2675454_1.fastq.gz PASS Per tile sequence quality ERR2675454_1.fastq.gz PASS Per sequence quality scores ERR2675454_1.fastq.gz WARN Per base sequence content ERR2675454_1.fastq.gz PASS Per sequence GC content ERR2675454_1.fastq.gz PASS Per base N content ERR2675454_1.fastq.gz PASS Sequence Length Distribution ERR2675454_1.fastq.gz FAIL Sequence Duplication Levels ERR2675454_1.fastq.gz PASS Overrepresented sequences ERR2675454_1.fastq.gz FAIL Adapter Content ERR2675454_1.fastq.gz ``` {% endcode %} {% code title="fastqc/ERR2675454\_1\_fastqc/fastqc\_data.txt" %} ```bash ##FastQC 0.11.9 >>Basic Statistics pass #Measure Value Filename ERR2675454_1.fastq.gz File type Conventional base calls Encoding Sanger / Illumina 1.9 Total Sequences 30273560 Sequences flagged as poor quality 0 Sequence length 151 %GC 47 >>END_MODULE ``` {% endcode %} Per base sequence quality of `ERR2675454_1.fastq.gz` ![](/files/-M5XeZ4OmhrGLWAQLD3u) ## Adapter removal and trimming The trimming process is run with 6 threads (`threads=6`) and took about **1.6** hours to complete. ```bash $ mkdir trimmed for id in ${runname[@]}; do adapters=/home/USER/tools/bbmap/resources/adapters.fa fq1=fastqs/${id}_1.fastq.gz fq2=fastqs/${id}_2.fastq.gz trim1=trimmed/${id}_1.fastq.gz trim2=trimmed/${id}_2.fastq.gz log=trimmed/${id}.log bbduk.sh threads=6 in1=$fq1 in2=$fq2 out1=$trim1 out2=$trim2 \ ref=$adapters tbo tpe ktrim=r k=21 mink=9 hdist=1 \ qtrim=rl trimq=15 minlength=36 maxns=1 2> $log done ``` ```bash # BBDuk parameters tbo - trim adapters based on pair overlap detection using BBMerge tpe - trim both reads to the same length ktrim=r - once a reference kmer is matched in a read, that kmer and all the bases to the right will be trimmed, leaving only the bases to the left k=21 - Kmer length used for finding contaminants mink=9 - look for shorter kmers at read tips down to 9 hdist=1 - maximum Hamming distance for ref kmers qtrim=rl trimq=15 - quality-trim to Q15 using the Phred algorithm for both sides minlength=36 - discard reads shorter than 36 bp after trimming maxns=1 - discard reads with more Ns than 1 after trimming ``` ```bash $ cd trimmed $ ls ERR2675454_1.fastq.gz ERR2675461_1.fastq.gz ERR2675472_1.fastq.gz ERR2675479_1.fastq.gz ERR2675454_2.fastq.gz ERR2675461_2.fastq.gz ERR2675472_2.fastq.gz ERR2675479_2.fastq.gz ERR2675454.log ERR2675461.log ERR2675472.log ERR2675479.log ERR2675455_1.fastq.gz ERR2675464_1.fastq.gz ERR2675473_1.fastq.gz ERR2675480_1.fastq.gz ERR2675455_2.fastq.gz ERR2675464_2.fastq.gz ERR2675473_2.fastq.gz ERR2675480_2.fastq.gz ERR2675455.log ERR2675464.log ERR2675473.log ERR2675480.log ERR2675458_1.fastq.gz ERR2675465_1.fastq.gz ERR2675476_1.fastq.gz ERR2675481_1.fastq.gz ERR2675458_2.fastq.gz ERR2675465_2.fastq.gz ERR2675476_2.fastq.gz ERR2675481_2.fastq.gz ERR2675458.log ERR2675465.log ERR2675476.log ERR2675481.log ERR2675459_1.fastq.gz ERR2675468_1.fastq.gz ERR2675477_1.fastq.gz ERR2675484_1.fastq.gz ERR2675459_2.fastq.gz ERR2675468_2.fastq.gz ERR2675477_2.fastq.gz ERR2675484_2.fastq.gz ERR2675459.log ERR2675468.log ERR2675477.log ERR2675484.log ERR2675460_1.fastq.gz ERR2675469_1.fastq.gz ERR2675478_1.fastq.gz ERR2675485_1.fastq.gz ERR2675460_2.fastq.gz ERR2675469_2.fastq.gz ERR2675478_2.fastq.gz ERR2675485_2.fastq.gz ERR2675460.log ERR2675469.log ERR2675478.log ERR2675485.log ``` Generated log files contain information about the number of reads and bases removed and passed the trimming processing {% code title="trimmed/ERR2675454.log" %} ```bash Input: 60547120 reads 9142615120 bases. QTrimmed: 19033902 reads (31.44%) 366761527 bases (4.01%) KTrimmed: 26320518 reads (43.47%) 982152960 bases (10.74%) Trimmed by overlap: 3581100 reads (5.91%) 18700662 bases (0.20%) Low quality discards: 16230 reads (0.03%) 2046404 bases (0.02%) Total Removed: 831678 reads (1.37%) 1369661553 bases (14.98%) Result: 59715442 reads (98.63%) 7772953567 bases (85.02%) Time: 300.984 seconds. Reads Processed: 60547k 201.16k reads/sec Bases Processed: 9142m 30.38m bases/sec ``` {% endcode %} --- # Agent Instructions: Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. Perform an HTTP GET request on the current page URL with the `ask` query parameter: ``` GET https://ycl6.gitbook.io/guide-to-rna-seq-analysis/raw-read-processing/qc-and-trimming.md?ask= ``` The question should be specific, self-contained, and written in natural language. 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