I have a MeatG library, which was sequenced by two times. The first time, we got unpaired reads (i.e., unpaired.fastq); while we got interlaced forward and reverse paired-end reads (i.e., interlaced-paired.fastq) for the 2nd time. Since they are from the same library, I assembled them together and got the assembled scaffolds. Now I would like to align both of them (unpaired and interlaced paired reads) simultaneously to the assembled contigs using Bowtie2 version 2.3.4.1 to get one .sam file. Can Bowtie2 do this? Any suggestions are appreciated!
I tried the following two scripts, the results for both were same. So for the first script, only the paired reads are mapped.
bowtie2 -q --phred33 --end-to-end --sensitive -p 12 -I 0 -X 2000 --no-unal -x bowtie2-db/CBIW --interleaved interlaced-paired.fastq -U unpaired.fastq | samtools view -Sb - > output.bam
bowtie2 -q --phred33 --end-to-end --sensitive -p 12 -I 0 -X 2000 --no-unal -x bowtie2-db/CBIW --interleaved interlaced-paired.fastq | samtools view -Sb - > output.bam
See the align result:
Building a SMALL index
35388893 reads; of these:
35388893 (100.00%) were paired; of these:
11580370 (32.72%) aligned concordantly 0 times
23621928 (66.75%) aligned concordantly exactly 1 time
186595 (0.53%) aligned concordantly >1 times
11580370 pairs aligned concordantly 0 times; of these:
101972 (0.88%) aligned discordantly 1 time
11478398 pairs aligned 0 times concordantly or discordantly; of these:
22956796 mates make up the pairs; of these:
22513992 (98.07%) aligned 0 times
432094 (1.88%) aligned exactly 1 time
10710 (0.05%) aligned >1 times
68.19% overall alignment rate
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xiaozhongzhiping •
I don't think Bowtie2 can map paired- and single-end reads simultaneously, but you can merge both mappings with samtools. BWA can map map a mix of interleaved paired reads and single reads, if you concatenate both files and keep single reads at the end.
Use samtools sort to sort each bam by position, then samtools merge to merge the sorted bams into one.
If having a sorted bam is not important, you can just use samtools cat.
Thanks. I followed your suggestions using samtools sort and merge. It works well.
Bowtie2 can map paired- and single-end reads simultaneously:
Like this:
bowtie2 -x btInd -1 13_trimmed_R1.fastq.gz -2 13_trimmed_R2.fastq.gz -U 13_unpaired_1.fastq.gz,13_unpaired_2.fastq.gz -p 12 | samtools sort -@ 12 -T temp -o 13trimmed_sorted.bam
Works for me. (bowtie2 version 2.3.2)
I can confirm this works well:
bowtie2 -x $idx -1 test_1.fq -2 test_2.fq -U test_single.fq
20000 reads; of these:
10000 (50.00%) were paired; of these:
1362 (13.62%) aligned concordantly 0 times
5857 (58.57%) aligned concordantly exactly 1 time
2781 (27.81%) aligned concordantly >1 times
----
1362 pairs aligned concordantly 0 times; of these:
76 (5.58%) aligned discordantly 1 time
----
1286 pairs aligned 0 times concordantly or discordantly; of these:
2572 mates make up the pairs; of these:
2156 (83.83%) aligned 0 times
244 (9.49%) aligned exactly 1 time
172 (6.69%) aligned >1 times
10000 (50.00%) were unpaired; of these:
1285 (12.85%) aligned 0 times
5798 (57.98%) aligned exactly 1 time
2917 (29.17%) aligned >1 times
88.53% overall alignment rate
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I have forward reads that didn't require any trimming except for adapter sequence. but the reverse read file required trimming so i trimmed from 131bp to 100bp. now idk if i can align these forward 131bp and reverse 100bp reads on my reference usin bowtie2 ?