User:Timothee Flutre/Notebook/Postdoc/2013/12/01

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One-liners for high-throughput sequencing data In this document, I'll focus on paired-end reads using, as an example, data from a GBS (RAD-seq) experiment in grapevine. mapping softwares: see BWA, Bowtie, MOSAIK, etc; they (usually) take fastq files as input and return bam files as output IN1="reads_R1.fastq.gz" IN2="reads_R2.fastq.gz" OUT="alignments.bam" number of input pairs: there are 2,307,772 pairs and 4,615,544 reads nbLines=$(zcat $IN1 | wc -l); echo "scale=0; "${nbLines}"/4" | bc -l # 2307772 nbLines=$(zcat $IN2 | wc -l); echo "scale=0; "${nbLines}"/4" | bc -l # 2307772 understand one read pair in the Fastq format: zcat $IN1 | head -4 zcat $IN2 | head -4 understand one alignment in the SAM format: samtools view $OUT | head -1 | tr "\t" "\n" | nl -n ln global alignment statistics: samtools flagstat $OUT which returns something like: line 1: 4635834 + 0 in total (QC-passed reads + QC-failed reads) line 2: 20290 + 0 secondary line 3: 0 + 0 supplimentary line 4: 0 + 0 duplicates line 5: 4443270 + 0 mapped (95.85%:-nan%) line 6: 4615544 + 0 paired in sequencing line 7: 2307772 + 0 read1 line 8: 2307772 + 0 read2 line 9: 4299122 + 0 properly paired (93.14%:-nan%) line 10: 4412810 + 0 with itself and mate mapped line 11: 10170 + 0 singletons (0.22%:-nan%) line 12: 57898 + 0 with mate mapped to a different chr line 13: 44330 + 0 with mate mapped to a different chr (mapQ>=5) list occurring BAM flags: along with their number of occurrences samtools view $OUT | cut -f2 | sort | uniq -c | sort -k1,1nr -k2,2n # | awk '{sum+=$1}END{print sum}' # <- same as line 1 list of bits used to form the flags this website is useful to interpret flags for instance, if there are N entries with flag 99, there should also be N entries with flag 147; idem for 83 and 163; idem for 77 and 141; etc entries in the BAM file: same as line 1; I use "entries" instead of "alignments" because the BAM file can contain unmapped reads (see below) samtools view -c $OUT # 4635834 mapped entries: same as line 5 samtools view -F 0x0004 -c $OUT # 4443270 echo "scale=3; 100 * 4443270 / 4635834" | bc -l # 95.846% unmapped entries: same as line 1 - line 5 samtools view -f 0x0004 -c $OUT # 192564 pairs in the BAM file: should be equal to the nb of input pairs samtools view $OUT | cut -f1 | sort | uniq | wc -l # 2307772 not-primary/supplementary entries: see details samtools view -f 0x0100 -c $OUT # 20290 <- same as line 2 samtools view -f 0x0800 -c $OUT # 0 <- same as line 3 R1 and R2 entries: nbE1=$(samtools view -f 0x0040 -c $OUT) # 2316858 nbE2=$(samtools view -f 0x0080 -c $OUT) # 2318976 echo "$nbE1 + $nbE2" | bc -l # 4635834 <- same as line 1 R1 and R2 reads: samtools view -f 0x0040 $OUT | cut -f1 | sort | uniq -c > tmp1 wc -l < tmp1 # 2307772 <- same as line 7 samtools view -f 0x0080 $OUT | cut -f1 | sort | uniq -c > tmp2 wc -l < tmp2 # 2307772 <- same as line 8 echo "$(wc -l < tmp1) + $(wc -l < tmp2)" | bc -l # 4615544 <- same as line 6 cat tmp1 | awk '{print $1}' | sort | uniq -c # some input reads can have multiple entries cat tmp2 | awk '{print $1}' | sort | uniq -c # idem entries properly paired in sequencing: irrespective of mapping samtools view -f 0x0001 -c $OUT # 4635834 <- same as line 1 if all input reads are paired samtools view -f 0x0001 -F 0x0100 -c $OUT # 4615544 <- same as line 6 entries properly paired in mapping: same as line 9 samtools view -f 0x0002 -F 0x0100 -c $OUT # 4299122 echo "scale=3; 100 * 4299122 / (4635834 - 20290)" | bc -l # 93.144% pairs, properly paired in mapping, in primary alignment: to be compared to the number of input pairs (see above) samtools view -f 0x0002 -F 0x0100 $OUT | cut -f1 | sort | uniq | wc -l # 2149561 echo "scale=3; 100 * 2149561 / 2307772" | bc -l # 93.144 singletons: same as line 11 samtools view -f 0x0008 -F 0x0004 -F 0x0100 -c $OUT # 10170 different chromosomes: same as lines 12 and 13 samtools view -F 0x0004 -F 0x0008 -F 0x0100 $OUT | cut -f7 | sort | uniq -c # to list all possible values the 7-th field is taking samtools view -F 0x0004 -F 0x0008 -F 0x0100 $OUT | awk '($7 != "=")' | wc -l # 57898 samtools view -q 5 -F 0x0004 -F 0x0008 -F 0x0100 $OUT | awk '($7 != "=")' | wc -l # 44330 strand: get all alignments on the reverse strand (use -F 0x10 for the forward) samtools view -f 0x10 $OUT | cut -f2 | sort | uniq -c samtools flags 113 # idem for 117, 121, etc