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--- archive:bioinformatic_tools:bwa [2011/05/16 19:27]
svohr
+++ archive:bioinformatic_tools:bwa [2011/05/23 02:25]
svohr [After SeqPrep]
@@ Line 28: / Line 28: @@
 bwa sampe database.fasta aln_sa1.sai aln_sa2.sai read1.fq read2.fq > aln.sam
 </code>
+===== Quirks =====
+The SAM formatted alignments include a column labeled "inferred insert length" by the BWA manual, but in the SAM specification it is described as the "template length" or distance between the leftmost mapped base to the rightmost mapped base. The second description seems to
+match the output of BWA. However, there are some template lengths that do not appear to be calculated correctly.
+<code>
+bc07_1.fastq:
+@HWUSI-EAS1722:4:66:6286:18215#CAGATC/1
+AGCAGTCGTCGTGGTATGCCTGGATGTTACAGCAGTCGTCGTGGTATGACTGGATGTTACAGCAGTCGTCGTGGTATGACTGGATGTTACAGCAGTCGTCGTGGTATGACTGGAT
+bc07_2.fastq:
+@HWUSI-EAS1722:4:66:6286:18215#CAGATC/2
+CACGACGACTGCTGTAACATCCAGGCATACCACGACGACTGCTGTAACATCCAGGCATACCACGACGACAGCTATAACATACACTCATACCACGA
+</code>
+For example, these two reads make up a pair that overlaps.
+<code>
+...ACATCCAGTCATACCACGACGACTGCTGTAACATCCAGGCATACCACGACGACTGCT
+                 CACGACGACTGCTGTAACATCCAGGCATACCACGACGACTGCTGTAACATCCAGGCATACCACGACGACAGCTATAACATACACTCATACCACGA
+</code>
+Instead of reporting the total length, the length of the overlap is reported.
+<code>
+HWUSI-EAS1722:4:66:6286:18215#CAGATC	81	GAZ7HUX03HIJAL	272	23	115M	=	344	-43
+HWUSI-EAS1722:4:66:6286:18215#CAGATC	161	GAZ7HUX03HIJAL	344	25	95M	=	272	43
+</code>
+This explains the incorrect short lengths found in our histograms. This does not appear to affect the pairs that do not overlap and most of these overlapping reads that should be combined using SeqPrep.
 ====== Determining Paired-End Insert Size ======
@@ Line 43: / Line 73: @@
 {{:bioinformatic_tools:run2_seqprep_template_size_histogram.png|}}
-Here is the same plot with the 454 length distribution for comparison.
-{{:bioinformatic_tools:run2_seqprep_template_size_histogram_with_454_length2.png|}}
+The next histogram shows the 454 length distribution with the SeqPrep merged read lengths and the mapped lengths for the paired-end templates. From this we can see that the lengths of the 454 reads are greater than the illumina templates and the short templates lengths are not due to the lengths of the sequences in the 454 reference.
+{{:bioinformatic_tools:run_all_illumina_v_454_histogram.png|}}
+These histograms show the mapped lengths for the paired-end templates and the lengths of merged reads from SeqPrep. In each of these, we can see the distinct range for the SeqPrep merged reads and the split between merged and unmerged pairs. The counts for the paired-end reads are lower than the counts for merged reads because the paired-end reads had to map to one of the 454 reads. We can see that if the paired-end counts were increased by a factor of 10 (for the 0.1x coverage in the 454 reads) the merged and paired-end reads would form a continuous distribution. Lengths less than 90 may be incorrect. The higher frequency of these in run 1 can be explained its higher coverage.
+In the merged lengths for both run 1 and run 2 barcode 8 there is a gap of 10 lengths (66-75 for run 1, 105-114 for run 2 bc08) where no reads were observed. This is an artifact of SeqPrep's two methods for merging reads and the 10 base overlap requirement for merging.
+{{:bioinformatic_tools:run1_seqprep_histogram_r2.png|}}
+{{:bioinformatic_tools:run2_bc07_seqprep_histogram_r2.png|}}
+{{:bioinformatic_tools:run2_bc08_seqprep_histogram_r2.png|}}

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